WO2012047317A2 - Anticorps spécifiques à une tumeur et utilisations de ceux-ci - Google Patents

Anticorps spécifiques à une tumeur et utilisations de ceux-ci Download PDF

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Publication number
WO2012047317A2
WO2012047317A2 PCT/US2011/037972 US2011037972W WO2012047317A2 WO 2012047317 A2 WO2012047317 A2 WO 2012047317A2 US 2011037972 W US2011037972 W US 2011037972W WO 2012047317 A2 WO2012047317 A2 WO 2012047317A2
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WIPO (PCT)
Prior art keywords
antibody
fragment
derivative
seq
cancer
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PCT/US2011/037972
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English (en)
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WO2012047317A3 (fr
Inventor
Pinku Mukherjee
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The University Of North Carolina At Charlotte
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Publication date
Priority to ES11831066T priority Critical patent/ES2927050T3/es
Priority to JP2013532795A priority patent/JP5886299B2/ja
Priority to CA2813814A priority patent/CA2813814C/fr
Priority to RU2013120483/10A priority patent/RU2595403C2/ru
Priority to CN201180059040.8A priority patent/CN103492417B/zh
Priority to KR1020137011650A priority patent/KR101883280B1/ko
Priority to BR112013008480A priority patent/BR112013008480A2/pt
Priority to MX2013003825A priority patent/MX346973B/es
Application filed by The University Of North Carolina At Charlotte filed Critical The University Of North Carolina At Charlotte
Priority to US13/877,582 priority patent/US9090698B2/en
Priority to AU2011312830A priority patent/AU2011312830B2/en
Priority to DK11831066.3T priority patent/DK2624866T3/da
Priority to EP11831066.3A priority patent/EP2624866B1/fr
Publication of WO2012047317A2 publication Critical patent/WO2012047317A2/fr
Publication of WO2012047317A3 publication Critical patent/WO2012047317A3/fr
Priority to IL225545A priority patent/IL225545A0/en
Priority to US14/810,209 priority patent/US20150368356A1/en
Priority to US14/991,145 priority patent/US9845362B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
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    • C—CHEMISTRY; METALLURGY
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    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
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    • C—CHEMISTRY; METALLURGY
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    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • G—PHYSICS
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Definitions

  • the presently disclosed subject matter relates to isolated antibodies, or fragments or derivatives thereof, which bind to antigens present in tumors, and methods of use therefor.
  • the presently disclosed subject matter relates to isolated antibodies, or fragments or derivatives thereof, that bind to the epithelial mucin family member MUC1 , and to methods for using the same to detect, target, and treat tumors and tumor cells, including but not limited to circulating tumor cells (CTCs) and cancer stem cells (CSCs).
  • CTCs circulating tumor cells
  • CSCs cancer stem cells
  • Pancreatic cancer is the fourth and fifth leading cause of cancer-related death for men and women, respectively, following lung, colon, and prostate cancers in men and lung, breast, colon, and ovarian cancers in women. Patients usually present with advanced disease, making treatment difficult. Surgery is the only curative therapy, yet local disease recurrence with or without spread to distant organs occurs in over 80% of patients. Attempts at better therapeutic modalities are necessary in order to improve outcome in this disease.
  • neoplastic transformation leads to alterations in the expression of various polypeptides in tumor cells.
  • certain mucins and mutated forms of K-ras oncogene polypeptides are overexpressed in 90% of pancreatic ductal adenocarcinomas (hereinafter referred to as "PDA"), and have been targets for therapeutic interventions.
  • PDA pancreatic ductal adenocarcinomas
  • vaccines that target these polypeptides have not been particularly successful clinically.
  • Vaccines have failed to generate long-term immune memory, likely due at least in part to tumors adapting in ways that allows them to escape immune recognition and killing.
  • Several agents that can modulate immune tolerance have been tested clinically, but with only modest responses, perhaps due to an insufficient amount of the agents reaching the tumor site and/or because the agents themselves have been associated with unwanted side effects such as can result from their binding to normal cells.
  • tumor stem cells frequently referred to as tumor stem cells or cancer stem cells
  • the presently disclosed subject matter provides the following:
  • Isolated antibodies as well as fragments and derivatives thereof, which specifically bind to mucin-1 (MUC1 ) and/or to mutated K-ras oncogene polypeptides present on epithelial tumors.
  • Isolated nucleic acids that encode the isolated antibodies of the presently disclosed subject matter and/or subsequences thereof.
  • Antibodies such as monoclonal antibodies, and/or peptides, fragments, and/or derivatives thereof, that bind specifically to tumors such as epithelial tumors, including pancreatic tumors, ovarian tumors, breast tumors, colorectal tumors, and metastatic lesions derived therefrom.
  • Antibodies as well as fragments and derivatives thereof, which bind specifically to an epitope present within a MUC1 polypeptide and/or a mutated K-ras G12D poypeptide, in some embodiments to an epitope present within a human MUC1 polypeptide.
  • the epitope is present within any of SEQ ID NOs: 1 -3.
  • Antibodies as well as fragments and derivatives thereof, which bind specifically to MUC1 (in some embodiments, human MUC1 ) and mutated K- ras G12D created using protein lysates from a mouse model which presents MUC1 and mutated K-ras G12D as tumor-associated antigens.
  • MUC1 in some embodiments, human MUC1
  • mutated K- ras G12D created using protein lysates from a mouse model which presents MUC1 and mutated K-ras G12D as tumor-associated antigens.
  • Chimeric molecules comprising antibodies, as well as fragments and derivatives thereof, attached to effectors and/or immune modulating agents, wherein the antibodies, or the fragments or derivatives thereof, bind specifically to epitopes present within a MUC1 polypeptide and/or a mutated K-ras polypeptide.
  • the effectors are selected from the group consisting of epitope tags, second antibodies (or fragments or derivatives thereof), labels, cytotoxins, liposomes, radionuclides, drugs, prodrugs, and chelates, and further wherein the immune modulating agent are selected from, for example, the agents listed in Table 3.
  • Antibodies as well as fragments and derivatives thereof, coupled to diagnostic agents.
  • Antibodies as well as fragments and derivatives thereof, prepared in compositions that comprise one or more pharmaceutically acceptable carriers and/or excipients.
  • Methods for inducing immune responses comprise introducing the antibodies, fragments, and/or derivatives thereof, and/or the compositions disclosed herein, into a subject such as, but not limited to, a human.
  • Methods for detecting cancerous cells comprising introducing into a subject such as but not limited to a human an antibody, or a fragment or derivative thereof, coupled to a detectable label that bind specifically to a MUC1 polypeptide and/or a mutated K-ras polypeptide.
  • Hybridoma cells that produce the antibodies, fragments, and/or derivatives of the presently disclosed subject matter, such as but not limited to monoclonal antibodies that bind specifically to a MUC1 polypeptide, a mutated K-ras G12D polypeptide, or both.
  • Vaccines against epithelial cancers comprising the antibodies, fragments, and/or derivatives of the presently disclosed subject matter and one or more pharmaceutically accepted carriers and/or excipients, optionally further comprising one orm roe immune modulating agents.
  • the presently disclosed subject matter provides isolated antibodies, or fragments or derivatives thereof, comprising the complementarity determining regions (CDRs) of monoclonal antibody TAB-004.
  • the isolated antibodies, or the fragments or derivatives thereof are polyclonal.
  • the isolated antibodies, or the fragments or derivatives thereof are monoclonal.
  • the isolated antibodies, or the fragments or derivatives thereof are human or humanized.
  • the isolated antibodies, or the fragments or derivatives thereof are selected from the group consisting of (a) a monoclonal antibody produced by hybridoma cell line TAB-004 deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 201 10-2209, United States of America, as Accession No.
  • ATCC American Type Culture Collection
  • the CDRs comprise one or more of the following: (i) heavy chain CDR1 comprises SEQ ID NO: 8; (ii) heavy chain CDR2 comprises SEQ ID NO: 9; (iii) heavy chain CDR3 comprises SEQ ID NO: 10; (iv) light chain CDR1 comprises SEQ ID NO: 1 1 ; (v) light chain CDR2 comprises SEQ ID NO: 12; and (vi) light chain CDR3 comprises SEQ ID NO: 13.
  • the heavy chain variable region comprises SEQ ID NO: 5 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 4; and/or the light chain variable region comprises SEQ ID NO: 7 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 6.
  • compositions comprising the presently disclosed antibodies, or the fragments or derivatives thereof, and one more pharmaceutically acceptable carriers and/or excipients.
  • the one orm ore pharmaceutically acceptable carriers and/or excipients are acceptable for use in a human.
  • compositions comprising the presently disclosed antibodies, or the fragments or derivatives thereof, conjugated to an active agent.
  • the active agent is selected from the group consisting of a radioactive molecule, a radionuclide, a sensitizer molecule, an imaging reagent, a radioisotope, a toxin, a cytotoxin, an anti-angiogenic agent, an anti-tumor agent, a chemotherapeutic agent, an immunomodulator, a cytokine, a reporter group, and combinations thereof.
  • the radioisotope is selected from the group consisting of 10 B, 211 At, 212 Pb, 212 Bi, 125 l, 3 1 , 35 S, and 3 H.
  • immunomodulator is selected from the group consisting of an indoleamine 2,3-dioxygenase (IDO) inhibitor, optionally, 1 -methyl-DL- tryptophan (1 MT); an EP2/EP4 receptor antagonist; a cyclooxygenase inhibitor, optionally indomethacin; and a dendritic cell activator, optionally CpG oligodeoxynucleotides (CpG ODN).
  • kits comprising the presently disclosed antibodies, or the fragments or derivatives thereof.
  • the presently disclosed kits comprise instructions for use of the presently disclosed antibodies, or the fragments or derivatives thereof.
  • the presently disclosed subject matter also provides delivery vehicles for use in targeted delivery of active agents to tumor cells.
  • the delivery vehicles comprise one orm ore targeting agents that comprise the presently disclosed antibodies, or the fragments or derivatives thereof.
  • the active agent comprises a radioactive molecule, a radionuclide, a sensitizer molecule, an imaging reagent, a radioisotope, a toxin, a cytotoxin, an anti-angiogenic agent, an antitumor agent, a chemotherapeutic agent, an immunomodulator, a cytokine, a reporter group, or a combination thereof.
  • the presently disclosed subject matter also provides isolated cells that produce the presently disclosed antibodies, or the fragments or derivative thereof.
  • the isolated cell is a hybridoma that produces the presently disclosed antibodies.
  • the hybridoma is hybridoma cell line ATCC Accession No. PTA-1 1550 deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 201 10-2209, United States of America, on December 16, 2010 under the terms of the Budapest Treaty.
  • the presently disclosed subject matter also provides methods for detecting the presence of an epitope to which monoclonal antibody TAB-004 binds in a biological sample.
  • the methods comprise: contacting the biological sample with one or more presently disclosed isolated antibodies, or fragments or derivatives thereof; and detecting the presence of an epitope to which monoclonal antibody TAB-004 binds in the biological sample.
  • the isolated antibodies, or the fragments or derivatives thereof bind to an epitope that is present within a mucin (MUC1 ) polypeptide and/or an epitope that is present within a K-ras polypeptide, optionally a mutant K-ras polypeptide.
  • MUC1 mucin
  • the presently disclosed subject matter also provides methods for making an antibody or a fragment or derivative thereof.
  • the methods comprise culturing an isolated cell of the presently disclosed subject matter or a hybridoma of the presently disclosed subject matter under conditions such that said antibody, the fragment, or the derivative thereof is expressed; and recovering the antibody or the fragment or derivative thereof from the cell or the hybridoma and/or from the environment in which the cell or the hybridoma is growing.
  • the presently disclosed subject matter also provides methods for detecting a tumor and/or a cancer cell in a subject.
  • the methods comprise contacting a biological sample in the subject or isolated from the subject with composition comprising a presently disclosed antibody, or a fragment or derivative thereof, under conditions sufficient for the presently disclosed antibody, or the fragment or derivative thereof, to bind to an epitope present on a tumor and/or a cancer cell, if present, in the biological sample; and detecting binding of the presently disclosed antibody, or the fragment or derivative thereof, to the epitope, wherein the detecting is indicative of a tumor and/or a cancer cell being present in the subject.
  • the tumor and/or the cancer cell is a tumor of the pancreas, breast, ovary, colon, or rectum, and/or a metastatic cell derived therefrom, which optionally expresses MUC1 , a mutant K-ras, or both.
  • the presently disclosed antibody, or the fragment or derivative thereof is conjugated to a detectable label comprising an imaging agent, which in some embodiments is selected from the group consisting of paramagnetic, radioactive, and fluorogenic ions.
  • the radioactive imaging agent is selected from the group consisting of gamma- emitters, positron-emitters and x-ray-emitters.
  • the radioactive imaging agent is selected from the group consisting of 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 77 Br, 81 Rb/ 81 MKr, 87M Sr, 99M Tc, 111 ln, 113 ln, 123 l, 125 l, 127 Cs, 129 Cs, 3 1, 132 l, 197 Hg, 203 Pb, and 206 Bi.
  • the biological sample is a blood sample, or a fraction derived therefrom.
  • the presently disclosed subject matter also provides methods for treating a tumor in a subject.
  • the methods comprise administering to the subject a composition comprising a presently disclosed antibody, or a fragment or derivative thereof, conjugated to an active agent, whereby the active agent contacts the tumor to thereby treat the tumor.
  • the active agent comprises a therapeutic agent, optionally a chemotherapeutic agent, a toxin, a radiotherapeutic agent, or a combination thereof.
  • the chemotherapeutic agent is selected from the group consisting of an anti-tumor drug, a cytokine, an antimetabolite, an alkylating agent, a hormone, methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, 5-fluorouracil, melphalan, chlorambucil, a nitrogen mustard, cyclophosphamide, cis-platinum, vindesine, vinca alkaloids, mitomycin, bleomycin, purothionin, macromomycin, 1 ,4- benzoquinone derivatives, trenimon, steroids, aminopterin, anthracyclines, demecolcine, etoposide, mithramycin, doxorubicin, daunomycin, vinblastine, neocarzinostatin, macromycin, a-amanitin, and combinations thereof.
  • the toxin is selected from the group consisting of Russell's Viper Venom, activated Factor IX, activated Factor X, thrombin, phospholipase C, cobra venom factor, ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, gelonin, saporin, modeccin, viscumin, volkensin, and combinations thereof.
  • the radiotherapeutic agent is selected from the group consisting of 47 Sc, 67 Cu, 90 Y, 109 Pd, 123 l, 125 l, 131 l, 186 Re, 188 Re, 199 Au, 211 At, 212 Pb, 212 Bi, 32 P, 33 P, 71 Ge, 77 As, 103 Pb, 105 Rh, 111 Ag, 119 Sb, 121 Sn, 131 Cs, 143 Pr, 161 Tb, 177 Lu, 191 Os, 193M Pt, and 197 Hg.
  • the presently disclosed subject matter also provides methods for suppressing tumor growth in a subject.
  • the methods comprise administering to a subject bearing a tumor an effective amount of an isolated antibody, or a fragment or derivative thereof, comprising the complementarity determining regions (CDRs) of monoclonal antibody TAB- 004.
  • the tumor is a tumor of the pancreas, breast, ovary, colon, or rectum, and/or a metastatic cell derived therefrom, which optionally expresses MUC1 , a mutant K-ras, or both.
  • the CDRs of TAB-004 comprise one or more of the following: heavy chain CDR1 comprises SEQ ID NO: 8; heavy chain CDR2 comprises SEQ ID NO: 9; heavy chain CDR3 comprises SEQ ID NO: 10; light chain CDR1 comprises SEQ ID NO: 1 1 ; light chain CDR2 comprises SEQ ID NO: 12; and/or light chain CDR3 comprises SEQ ID NO: 13.
  • the heavy chain variable region comprises SEQ ID NO: 5 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 4, and/or the light chain variable region comprises SEQ ID NO: 7 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 6.
  • the methods further comprise administering to the subject one or more additional anti-tumor treatments.
  • the one or more additional anti-tumor treatments are selected from the group consisting of radiotherapy, chemotherapy, an additional immunotherapy, an antiinflammatory therapy, and combinations thereof.
  • the anti-inflammatory therapy comprises administering to the subject a cyclooxygenase inhibitor, optionally a cyclooxygenase-2-specific inhibitor.
  • the one or more additional anti-tumor therapies comprise administering gemcitabine (4-amino-1 -(2-deoxy-2,2-difluoro-P-D-ery#7ro- pentofuranosyl)pyrimidin-2(1 H)-on-2',2'-difluoro-2'-deoxycytidine) and celecoxib (4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1 - yljbenzenesulfonamide), or pharmaceutically acceptable salts of either or both thereof, to the subject.
  • gemcitabine 4-amino-1 -(2-deoxy-2,2-difluoro-P-D-ery#7ro- pentofuranosyl)pyrimidin-2(1 H)-on-2',2'-difluoro-2'-deoxycytidine
  • celecoxib 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1
  • the presently disclosed subject matter also provides methods for purifying cancer stem cells.
  • the methods comprise providing a population of cells suspected of comprising cancer stem cells; identifying a subpopulation of the cells that bind to an antibody, or a fragment or derivative thereof, comprising the CDRs of monoclonal antibody TAB-004; and purifying the subpopulation.
  • the population of cells comprises circulating cells isolated from a subject that has a tumor and/or a cancer.
  • the presently disclosed methods further comprise removing lineage-positive (lin + ) cells from the population of cells before the identifying step and/or after the purifying step.
  • the presently disclosed subject matter also provides methods for targeting active agents to circulating cancer stem cells in subjects.
  • the methods comprise contacting the circulating cancer stem cells with a composition comprising a presently disclosed antibody, or a fragment or derivative thereof, comprising the CDRs of monoclonal antibody TAB-004 and one or more active agents, optionally wherein the one orm ore active agents comprise a therapeutic agent, optionally a chemotherapeutic agent, a toxin, a radiotherapeutic agent, or a combination thereof.
  • the therapeutic agent comprises an immunomodulator, optionally wherein the immunomodulator is selected from the group consisting of wherein the immunomodulator is selected from the group consisting of an indoleamine 2,3-dioxygenase (IDO) inhibitor, optionally, 1 -methyl-DL- tryptophan (1 MT); an EP2/EP4 receptor antagonist; and a dendritic cell activator, optionally CpG oligodeoxynucleotides (CpG ODN).
  • IDO indoleamine 2,3-dioxygenase
  • MT 1 -methyl-DL- tryptophan
  • EP2/EP4 receptor antagonist an EP2/EP4 receptor antagonist
  • a dendritic cell activator optionally CpG oligodeoxynucleotides (CpG ODN).
  • the presently disclosed subject matter also provides methods for prognosing recurrence of cancer in a subject previously treated for the cancer.
  • the methods comprise isolating a biological sample comprising circulating cells from a subject with a cancer; contacting the biological sample with the presently disclosed antibodies, or the fragments or derivatives thereof, under conditions sufficient for the antibodies, or the fragments or derivatives thereof, to bind to an epitope present on a tumor and/or a cancer cell, if present, in the biological sample; and identifying in the biological sample one or more circulating cells that bind to the antibody, or the fragment or derivative thereof, whereby recurrence of a cancer is prognosed in the subject.
  • the biological sample comprises a blood sample, a lymph sample, or a fraction thereof.
  • the cancer is a pancreatic cancer or a breast cancer.
  • the antibodies, or the fragments or derivatives thereof are selected from the group consisting of monoclonal antibodies produced by hybridoma cell line TAB-004 deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 201 10-2209, United States of America, on December 16, 2010 under the terms of the Budapest Treaty, as Accession No.
  • PTA-1 1550 chimeric antibodies, or fragments or derivatives thereof; humanized antibodies, or fragments or derivatives thereof; human antibodies, or fragments or derivatives thereof; single chain antibodies, or fragments or derivatives thereof; and Fab fragments, and further wherein the chimeric antibodies, the humanized antibodies, the human antibodies, the single chain antibodies, or the Fab fragments comprise the complementarity determining regions (CDRs) of monoclonal antibody TAB-004.
  • CDRs complementarity determining regions
  • the CDRs of monoclonal antibody TAB-004 comprise the following amino acid sequences: heavy chain CDR1 comprises SEQ ID NO: 8; heavy chain CDR2 comprises SEQ ID NO: 9; heavy chain CDR3 comprises SEQ ID NO: 10; light chain CDR1 comprises SEQ ID NO: 1 1 ; light chain CDR2 comprises SEQ ID NO: 12; and light chain CDR3 comprises SEQ ID NO: 13.
  • the heavy chain variable region comprises SEQ ID NO: 5 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 4; and/or the light chain variable region comprises SEQ ID NO: 7 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 6.
  • the presently disclosed subject matter also provides methods for prognosing progression of a cancer in a subject.
  • the methods comprise isolating a biological sample comprising circulating cells from a subject with a cancer; contacting the biological sample with an antibody, or a fragment or derivative thereof, of the presently disclosed subject matter under conditions sufficient for the antibody, or the fragment or derivative thereof, to bind to an epitope present on a tumor and/or a cancer cell, if present, in the biological sample; and identifying in the biological sample one or more circulating cells that bind to the antibody, or the fragment or derivative thereof, whereby progression of a cancer is prognosed in the subject.
  • the biological sample comprises a blood sample, a lymph sample, or a fraction thereof.
  • the cancer is a pancreatic cancer or a breast cancer.
  • the antibody, or the fragment or derivative thereof is selected from the group consisting of a monoclonal antibody produced by hybridoma cell line TAB-004 deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 201 10-2209, United States of America, on December 16, 2010 under the terms of the Budapest Treaty as Accession No.
  • a chimeric antibody, or a fragment or derivative thereof comprises the complementarity determining regions (CDRs) of monoclonal antibody TAB- 004.
  • the CDRs of monoclonal antibody TAB-004 comprise the following amino acid sequences: heavy chain CDR1 comprises SEQ ID NO: 8; heavy chain CDR2 comprises SEQ ID NO: 9; heavy chain CDR3 comprises SEQ ID NO: 10; light chain CDR1 comprises SEQ ID NO: 1 1 ; light chain CDR2 comprises SEQ ID NO: 12; and light chain CDR3 comprises SEQ ID NO: 13.
  • the heavy chain variable region comprises SEQ ID NO: 5 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 4; and/or the light chain variable region comprises SEQ ID NO: 7 or is encoded by a nucleic acid molecule comprising SEQ ID NO: 6.
  • progression of the cancer comprises metastasis of the cancer in the subject.
  • the presently disclosed subject matter also provides isolated nucleic acid molecules comprising any of SEQ ID ID NOs: 4 and 6, and/or encoding any of SEQ ID NOs: 5 and 7-13.
  • the isolated nucleic acid molecule is present within a vector, which in some embodiments is an expression vector.
  • the isolated nucleic acid molecule is present within an expression vector operably linked to one or more additional nucleotide sequences encoding subsequences of antibody molecules such that upon introduction of the expression vector into an appropriate host, an intact recombinant antibody comprising one or more of SEQ ID NOs: 5 and 7-13, or a fragment or derivative thereof, is expressed by the host cell.
  • it is an object of the presently disclosed subject matter to provide isolated antibodies, and fragments and derivatives thereof, which bind to antigens present in tumors.
  • Figures 1A and 1 B are a series of photomicrographs depicting specific binding of an exemplary antibody of the presently disclosed subject matter to human and mouse pancreatic tumors. The dark staining in the panels is indicative of positive binding of the exemplary antibody to cells present in the sample.
  • Figure 1A is a series of photomicrographs depicting the binding of the exemplary antibody to human tumors at Stages 0 (normal pancreatic tissue as a negative control) and 2-4.
  • Figure 1 B is a series of photomicrographs depicting the binding of an exemplary antibody of the presently disclosed subject matter to spontaneous tumors present in the pancreas of 6, 16, 26, and 34 week old transgenic mice that carried a human MUC1 transgene and a K-ras G12D mutation.
  • Figures 2A-2C are a series of photomicrographs depicting specific binding of an exemplary antibody of the presently disclosed subject matter to human breast tumor tissue ( Figures 2A and 2B) but not to adjacent normal breast tissue ( Figure 2C).
  • FIG 3 is a schematic (upper left panel) showing an approach for creating a triple transgenic mouse line that expresses the Cre recombinase throughout the pancreas, a mutated K-ras oncogene polypeptide, and a human MUC1 polypeptide.
  • This mouse develops pancreatic adenocarcinoma, cells of which were used to generate the primary tumor cell line KCM (lower panel).
  • the KCM cell line was used to test the binding of an exemplary antibody of the presently disclosed subject matter (denoted in the Figure as "TAB” and in the instant disclosure as "TAB-004") either alone or conjugated to CpG oligodeoxynucleotides (CpG ODN). It was determined that both the unconjugated and conjugated antibodies bound to the KCM pancreatic cell line with equal affinity (upper right panel).
  • Figures 4A and 4B are graphs showing that an exemplary antibody of the presently disclosed subject matter (TAB-004) enhanced the cytotoxicity of Natural Killer (NK) cells to kill target tumor cells. Conjugation of the antibody to CpG ODN further enhanced this effect, thereby demonstrating that the exemplary antibody was capable of enhancing an anti-tumor immune response in vivo.
  • TAB-004 an exemplary antibody of the presently disclosed subject matter
  • NK Natural Killer
  • Figure 4A is a line graph showing that the exemplary TAB-004 antibody enhanced specific lysis of KCM tumor cells at various effector (NK cells) to target ratios (E:T ratio) relative to a negative control (minus TAB-004 antibody).
  • Figure 4B is a bar graph showing that conjugation of the exemplary TAB-004 antibody to CpG ODN further enhanced specific lysis of tumor cells at various E:T ratios relative to the unconjugated antibody.
  • Figures 5A and 5B illustrate the results of experiments designed to test the ability of an exemplary antibody of the presently disclosed subject matter (TAB-004), and conjugates thereof, to reduce tumor volume of an established KCM tumor in MUC1 transgenic (MUC1 Tg) mice.
  • Figure 5A is a line graph showing the measured tumor volumes (in mm measured by digital calipers) in mice treated with phosphate-buffered saline (PBS) alone (negative control; filled squares), CpG ODN alone (X's), the unconjugated TAB-004 antibody (open circles), or the TAB-004-CpG ODN conjugate (filled circles).
  • PBS phosphate-buffered saline
  • X's CpG ODN alone
  • X's the unconjugated TAB-004 antibody
  • TAB-004-CpG ODN conjugate filled circles
  • FIG. 5B is a bar graph showing the changes in tumor volumes in mice at 19 and 27 days after the final treatment was administered.
  • the tumor volumes in the mice treated with TAB-004-CpG ODN had not increased as compared to the control mice (i.e., PBS alone, CpG ODN alone, or TAB-004 alone).
  • Figure 6 is a schematic depiction of exemplary compositions of the presently disclosed subject matter and exemplary uses therefor. ADCC - antibody dependent cell-mediated cytotoxicity; CDC - complement dependent cytotoxicity; ADEPT - antibody directed enzyme prodrug therapy.
  • Figures 7A and 7B are histograms of fluorescence-activated cell sorting (FACS) separations of CD133 + ( Figure 7A) versus CD24 + /CD447EpCAM + ( Figure 7B) cells and the extent to which the TAB-004 antibody disclosed herein bound to these populations.
  • the left trace in each panel corresponds to sorting with a negative control antibody, and the right trace in each panel corresponds to sorting with the TAB-004 antibody.
  • FIGS 8A-8D are FACS scatter plots showing binding of the TAB-004 antibody to a pancreatic tumor ("Tumorl ”) and adjacent normal tissue (“Normal”) using the TAB-004 antibody and an antibody directed against the CXC chemokine receptor 4 (CXCR4).
  • MUC1 TAB-004 antibody
  • CXCR4 anti-CXCR4 antibody
  • FL1 -H Flourescent stain 1 height (Flourescein-FITC);
  • FL2-H Flourescent stain 2 height (Phycoerythrin-PE);
  • SSC-H side-scatter height;
  • FSC-H forward-scatter height; FL4-H: Flourescent stain 4 - height (Allophycocyanin-APC).
  • Figure 8A is a scatter plot showing the distributions of cells in the absence of either antibody.
  • Figure 8B is a scatter plot showing the distributions of cells stained with an isotype control.
  • Figure 8C is a series of scatter plots showing the distributions of cells in normal tissue stained with the TAB-004 antibody versus a CXCR4 antibody.
  • Figure 8D is a series of scatter plots showing the distributions of cells in pancreatic adenocarcinoma tissue stained with the TAB-004 antibody versus a CXCR4 antibody.
  • FIGS 9A-9C are a series of FACS plots that show that the TAB-004 antibody of the presently disclosed subject matter is superior to a standard EpCAM antibody in detecting circulating tumor cells in pancreatic cancer patients.
  • Unstained cells left most black lines; EpCAM-PE (0.1 mg/ml) stained cells (dark gray lines); TAB-004-PE (0.1 mg/ml) stained cells (right most black lines); TAB-004-PE (0.02 mg/ml) stained cells (light gray lines); TAB-004-PE (0.004 mg/ml) stained cells (medium gray lines).
  • "-PE” indicates that the antibodies were labeled with phycoerythrin for the purposes of sorting.
  • FIG 9A PANC1 pancreatic cancer cell line cells were detected by the TAB-004-PE antibody.
  • Figures 9B and 9C circulating tumor cells present in the blood from two patients (patient number 1 and patient number 2, respectively) were detected by the TAB-004-PE antibody (see the right most black line in Figure 9B and the right most black and light gray lines in Figure 9C) but not the EpCAM-PE antibody (see the light gray lines in Figures 9B and 9C) that is currently in use.
  • Figure 10 is a bar graph of a comparison of performance of an antibody specific for the CA 15-3 antigen with TAB-004 in an enzyme immunoassay (EIA) for detecting cancer cells in plasma.
  • EIA enzyme immunoassay
  • Figure 1 1 is a bar graph of a comparison of performance of TAB-004 in an enzyme immunoassay (EIA) fopr detecting levels of shed MUC1 in plasma of pancreatic patients as a function of stage compared to a CA15-3-based EIA.
  • EIA enzyme immunoassay
  • SEQ ID NO: 1 is an amino acid sequence of a human MUC1 gene product. It corresponds to GENBANK® Accession No. AAA60019.
  • SEQ ID NO: 2 is an amino acid sequence of a human K-ras oncogene product. It corresponds to GENBANK® Accession No. NP_004976.
  • SEQ ID NO: 3 is an amino acid sequence of peptide to which the TAB- 004 antibody disclosed herein can bind in an ELISA assay.
  • the peptide thus includes an epitope to which the TAB-004 antibody binds specifically.
  • SEQ ID Nos: 4 and 5 are the nucleotide and encoded amino acid sequences, respectively, of the heavy chain of the TAB-004 antibody disclosed herein.
  • SEQ ID NOs: 6 and 7 are the nucleotide and encoded amino acid sequences, respectively, of the light chain of the TAB-004 antibody disclosed herein.
  • SEQ ID NOs: 8-10 are the amino acid sequences of CDR1 , CDR2, and CDR3, respectively, of the heavy chain of the TAB-004 antibody disclosed herein.
  • SEQ ID NOs: 1 1 -13 are the amino acid sequences of CDR1 , CDR2, and CDR3, respectively, of the light chain of the TAB-004 antibody disclosed herein.
  • the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including in the claims.
  • the phrase “an antibody” refers to one or more antibodies, including a plurality of the same antibody.
  • the phrase “at least one”, when employed herein to refer to an entity refers to, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to whole number values between 1 and 100 and greater than 100.
  • the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
  • a pharmaceutical composition can "consist essentially of a pharmaceutically active agent or a plurality of pharmaceutically active agents, which means that the recited pharmaceutically active agent(s) is/are the only pharmaceutically active agent(s) present in the pharmaceutical composition. It is noted, however, that carriers, excipients, and/or other inactive agents can and likely would be present in such a pharmaceutical composition.
  • compositions comprising antibodies. It would be understood by one of ordinary skill in the art after review of the instant disclosure that the presently disclosed subject matter thus encompasses compositions that consist essentially of the antibodies of the presently disclosed subject matter, as well as compositions that consist of the antibodies of the presently disclosed subject matter.
  • subject refers to a member of any invertebrate or vertebrate species.
  • the term "subject” is intended to encompass in some embodiments any member of the Kingdom Animalia including, but not limited to the phylum Chordata (e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all Orders and Families encompassed therein.
  • phylum Chordata e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all Orders and Families encompassed therein.
  • compositions and methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates.
  • the presently disclosed subject matter concerns mammals and birds. More particularly provided are compositions and methods derived from and/or for use in mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, and rabbits), marsupials, and horses.
  • carnivores other than humans such as cats and dogs
  • swine pigs, hogs, and wild boars
  • domesticated fowl e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans.
  • livestock including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs and/or orthologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • the human amino acid sequences disclosed are intended to encompass homologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. Also encompassed are any and all nucleotide sequences that encode the disclosed amino acid sequences, including but not limited to those disclosed in the corresponding GENBANK® entries (i.e., J05582.1 and NM_004985, respectively).
  • cancer and “tumor” are used interchangeably herein and can refer to both primary and metastasized solid tumors and carcinomas of any tissue in a subject, including but not limited to breast; colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bile ducts; small intestine; urinary tract including kidney, bladder, and urothelium; female genital tract including cervix, uterus, ovaries (e.g.
  • choriocarcinoma and gestational trophoblastic disease male genital tract including prostate, seminal vesicles, testes and germ cell tumors; endocrine glands including thyroid, adrenal, and pituitary; skin (e.g., hemangiomas and melanomas), bone or soft tissues; blood vessels (e.g. , Kaposi's sarcoma); brain, nerves, eyes, and meninges (e.g. , astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas and meningiomas).
  • skin e.g., hemangiomas and melanomas
  • blood vessels e.g. , Kaposi's sarcoma
  • brain nerves, eyes, and meninges (e.g. , astrocytomas, gliomas, glioblastomas, retinoblastomas, neuro
  • a cancer or a tumor comprises a cancer or tumor of an epithelial tissue such as, but not limited to a carcinoma.
  • a tumor is an adenocarcinoma, which in some embodiments is an adenocarcinoma of the pancreas, breast, ovary, colon, or rectum, and/or a metastatic cell derived therefrom.
  • effector refers to any molecule or combination of molecules whose activity it is desired to deliver/into and/or localize at a cell. Effectors include, but are not limited to labels, cytotoxins, enzymes, growth factors, transcription factors, drugs, etc.
  • effector refers to an immune system cell that can be induced to perform a specific function associated with an immune response to a stimulus.
  • exemplary effector cells include, but are not limited to natural killer (NK) cells and cytotoxic T cells (T c cells).
  • expression vector refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the construct comprising the nucleotide sequence of interest can be chimeric. The construct can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression vector comprises an isolated nucleic acid molecule of the presently disclosed subject matter, which in some embodiments comprises any of SEQ ID ID NOs: 4 and 6, or encodes any of SEQ ID NOs: 5 and 7-13.
  • the isolated nucleic acid molecule present within an expression vector is operably linked to one or more additional nucleotide sequences encoding subsequences of antibody molecules such that upon introduction of the expression vector into an appropriate host, an intact recombinant antibody comprising one or more of SEQ ID NOs: 5 and 7-13, or a fragment or derivative thereof, is expressed by the host cell.
  • hybridoma refers to a cell or cell line that is produced in the laboratory from the fusion of an antibody-producing lymphocyte and a non-antibody-producing cancer cell, usually a myeloma or lymphoma cell.
  • a hybridoma can proliferate and produce a continuous supply of a specific monoclonal antibody. Methods for generating hybridomas are known in the art (see e.g., Harlow & Lane, 1988).
  • operatively linked and “operably linked refer to transcriptional regulatory elements (such as, but not limited to promoter sequences, transcription terminator sequences, etc.) that are connected to a nucleotide sequence (for example, a coding sequence or open reading frame) in such a way that the transcription of the nucleotide sequence is controlled and regulated by that transcriptional regulatory element.
  • a nucleotide sequence is said to be under the "transcriptional control" of a promoter to which it is operably linked.
  • prodrug refers to an analog and/or a precursor of a drug (e.g., a cytotoxic agent) that substantially lacks the biological activity of the drug (e.g., a cytotoxic activity) until subjected to an activation step.
  • Activation steps can include enzymatic cleavage, chemical activation steps such as exposure to a reductant, and/or physical activation steps such as photolysis. In some embodiments, activiation occurs in vivo within the body of a subject,
  • the presently disclosed subject matter provides in some embodiments isolated antibodies, as well as fragments and derivatives thereof, which bind to antigens present within tumors such as, but not limited to antigens present within MUC1 polypeptides.
  • antibody and “antibodies” refer to proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • Immunoglobulin genes typically include the kappa ( ⁇ ), lambda ( ⁇ ), alpha (a), gamma ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), and mu ( ⁇ ) constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either ⁇ or ⁇ . In mammals, heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the term “antibody” refers to an antibody that binds specifically to an epitope that is present on a tumor antigen including, but not limited to a MUC1 polypeptide and/or a mutant K-ras polypeptide. In some embodiments, the term “antibody” refers to an antibody that binds specifically to an epitope present within any of SEQ ID NOs: 1 -3.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” chain (average molecular weight of about 25 kiloDalton (kDa)) and one "heavy” chain (average molecular weight of about 50-70 kDa).
  • the two identical pairs of polypeptide chains are held together in dimeric form by disulfide bonds that are present within the heavy chain region.
  • the N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition (sometimes referred to as the "paratope”).
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.
  • Antibodies typically exist as intact immunoglobulins or as a number of well-characterized fragments that can be produced by digestion with various peptidases. For example, digestion of an antibody molecule with papain cleaves the antibody at a position N-terminal to the disulfide bonds. This produces three fragments: two identical "Fab” fragments, which have a light chain and the N-terminus of the heavy chain, and an "Fc" fragment that includes the C-terminus of the heavy chains held together by the disulfide bonds.
  • Pepsin digests an antibody C-terminal to the disulfide bond in the hinge region to produce a fragment known as the "F(ab)'2" fragment, which is a dimer of the Fab fragments joined by the disulfide bond.
  • the F(ab)' 2 fragment can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab') 2 dimer into two "Fab"' monomers.
  • the Fab' monomer is essentially an Fab fragment with part of the hinge region (see e.g., Paul, 1993, for a more detailed description of other antibody fragments).
  • Fab, F(ab') 2 , and Fab' fragments include at least one intact antigen binding domain (paratope), and thus are capable of binding to antigens. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that various of these fragments (including, but not limited to Fab' fragments) can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term “antibody” as used herein also includes antibody fragments produced by the modification of whole antibodies and/or synthesized de novo using recombinant DNA methodologies. In some embodiments, the term “antibody” comprises a fragment that has at least one antigen binding domain (paratope).
  • Antibodies can be polyclonal or monoclonal. As used herein, the term
  • polyclonal refers to antibodies that are present together in a given collection of antibodies and that are derived from different antibody-producing cells (e.g., B cells).
  • Exemplary polyclonal antibodies include, but are not limited to those antibodies that bind to a particular antigen and that are found in the blood of an animal after that animal has produced an immune response against the antigen.
  • a polyclonal preparation of antibodies can also be prepared artificially by mixing at least non-identical two antibodies.
  • polyclonal antibodies typically include different antibodies that are directed against (i.e., bind to) the same and/or different epitopes (sometimes referred to as an "antigenic determinant" or just "determinant”) of any given antigen.
  • the term “monoclonal” refers to a single antibody species and/or a substantially homogeneous population of a single antibody species. Stated another way, “monoclonal” refers to individual antibodies or populations of individual antibodies in which the antibodies are identical in specificity and affinity except for possible naturally occurring mutations that can be present in minor amounts. Typically, a monoclonal antibody (mAb or moAb) is generated by a single B cell or a progeny cell thereof (although the presently disclosed subject matter also encompasses “monoclonal” antibodies that are produced by molecular biological techniques as described herein). Monoclonal antibodies (mAbs or moAbs) are highly specific, typically being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, a given mAb is typically directed against a single epitope on the antigen.
  • mAbs can be advantageous for some purposes in that they can be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method, however.
  • the mAbs of the presently disclosed subject matter are prepared using the hybridoma methodology first described by Kohler et al., 1975, and in some embodiments are made using recombinant DNA methods in prokaryotic or eukaryotic cells (see e.g. , U.S. Patent No. 4,816,567, the entire contents of which are incorporated herein by reference).
  • mAbs can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., 1991 and Marks et al., 1991 .
  • the antibodies, fragments, and derivatives of the presently disclosed subject matter can also include chimeric antibodies.
  • chimeric refers to antibody derivatives that have constant regions derived substantially or exclusively from antibody constant regions from one species and variable regions derived substantially or exclusively from the sequence of the variable region from another species.
  • variable region allows an antibody to selectively recognize and specifically bind epitopes on antigens. That is, the V L domain and V H domain, or subsets of the complementarity determining regions (CDRs) within these variable domains, of an antibody combine to form the variable region that defines a three dimensional antigen binding site.
  • This quaternary antibody structure forms the antigen binding site present at the end of each arm of the antibody. More specifically, the antigen binding site is defined by three CDRs on each of the V H and V L chains.
  • a complete immunoglobulin molecule can consist of heavy chains only with no light chains (see e.g., Hamers- Casterman et al., 1993).
  • CDRs present in each antigen binding domain that are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen binding domains referred to as "framework" regions, show less inter-molecular variability.
  • the framework regions largely adopt a ⁇ -sheet conformation and the CDRs form loops that connect, and in some cases form part of, the ⁇ - sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable domain by one of ordinary skill in the art, since they have been precisely defined (see e.g., Chothia & Lesk, 1987; Kabat et al., 1991 ; Martin, 1996; Johnson & Wu, 2000).
  • a particular kind of chimeric antibody is a "humanized" antibody, in which the antibodies are produced by substituting the CDRs of, for example, a mouse antibody, for the CDRs of a human antibody (see e.g., PCT International Patent Application Publication No. WO 1992/22653).
  • a humanized antibody has constant regions and variable regions other than the CDRs that are derived substantially or exclusively from the corresponding regions of a human antibody, and CDRs that are derived substantially or exclusively from a mammal other than a human.
  • the antibodies, fragments, and derivatives of the presently disclosed subject matter can also be single chain antibodies and single chain antibody fragments.
  • Single-chain antibody fragments contain amino acid sequences having at least one of the variable regions and/or CDRs of the whole antibodies described herein, but are lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of whole antibodies.
  • Single-chain antibody fragments can overcome some of the problems associated with the use of antibodies containing a part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, and/or other unwanted biological activities. Additionally, single-chain antibody fragments are considerably smaller than whole antibodies and can therefore be characterized by greater capillary permeability than whole antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Also, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely than whole antibodies to provoke an immune response in a recipient.
  • the single-chain antibody fragments of the presently disclosed subject matter include, but are not limited to single chain fragment variable (scFv) antibodies and derivatives thereof such as, but not limited to tandem di-scFv, tandem tri-scFv, diabodies, triabodies, tetrabodies, miniantibodies, and minibodies.
  • scFv single chain fragment variable
  • Fv fragments correspond to the variable fragments at the N-termini of immunoglobulin heavy and light chains. Fv fragments appear to have lower interaction energy of their two chains than Fab fragments. To stabilize the association of the V H and V L domains, they can be linked with peptides (see e.g. , Bird et al., 1988; Huston et al. , 1988), disulfide bridges (see e.g. , Glockshuber et al., 1990), and/or "knob in hole” mutations (see e.g. , Zhu et al., 1997). ScFv fragments can be produced by methods well known to those skilled in the art (see e.g. , Whitlow et al., 1991 ; Huston et al., 1993).
  • scFv can be produced in bacterial cells such as E. coli or in eukaryotic cells.
  • One potential disadvantage of scFv is the monovalency of the product, which can preclude an increased avidity due to polyvalent binding, and their short half-life.
  • Attempts to overcome these problems include bivalent (scFv') 2 produced from scFv containing an additional C-terminal cysteine by chemical coupling (see e.g. , Adams et al. , 1993; McCartney et al., 1995) or by spontaneous site-specific dimerization of scFv containing an unpaired C- terminal cysteine residue (see e.g., Kipriyanov et al., 1995).
  • scFv can be forced to form multimers by shortening the peptide linker to 3 to 12 residues to form "diabodies" (see e.g., Holliger et al., 1993). Reducing the linker still further can result in scFv trimers ("triabodies”; see e.g., Kortt et al., 1997) and tetramers ("tetrabodies”; see e.g., Le Gall et al., 1999).
  • scFv-scFv tandems ((scFv) 2 ) can be produced by linking two scFv units by a third peptide linker (see e.g., Kurucz et al., 1995).
  • Bispecific diabodies can be produced through the non-covalent association of two single chain fusion products consisting of V H domain from one antibody connected by a short linker to the V L domain of another antibody (see e.g., Kipriyanov et al., 1998).
  • the stability of such bispecific diabodies can be enhanced by the introduction of disulfide bridges or "knob in hole” mutations as described hereinabove or by the formation of single chain diabodies (scDb) wherein two hybrid scFv fragments are connected through a peptide linker (see e.g., Kontermann et al., 1999).
  • Tetravalent bispecific molecules can be produced, for example, by fusing an scFv fragment to the CH 3 domain of an IgG molecule or to a Fab fragment through the hinge region (see e.g., Coloma et al., 1997).
  • tetravalent bispecific molecules have been created by the fusion of bispecific single chain diabodies (see e.g., Alt et al., 1999).
  • Smaller tetravalent bispecific molecules can also be formed by the dimerization of either scFv-scFv tandems with a linker containing a helix-loop-helix motif (DiBi miniantibodies; see e.g., Muller et al., 1998) or a single chain molecule comprising four antibody variable domains (V H and V L ) in an orientation preventing intramolecular pairing (tandem diabody; see e.g., Kipriyanov et al., 1999).
  • Bispecific F(ab') 2 fragments can be created by chemical coupling of Fab' fragments or by heterodimerization through leucine zippers (see e.g., Shalaby et al., 1992; Kostelny et al., 1992). Also available are isolated V H and V L domains (see U.S. Patent Nos. 6,172,197; 6,248,516; and 6,291 , 158).
  • the presently disclosed subject matter also includes functional equivalents of the antibodies of the presently disclosed subject matter.
  • the phrase "functional equivalent” as it refers to an antibody refers to a molecule that has binding characteristics that are comparable to those of a given antibody.
  • chimerized, humanized, and single chain antibodies, as well as fragments thereof are considered functional equivalents of the corresponding antibodies upon which they are based.
  • the presently disclosed subject matter provides functional equivalents of the TAB-004 mAb disclosed herein.
  • Functional equivalents also include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the presently disclosed subject matter.
  • the phrase "substantially the same” refers to a biosequence with in some embodiments at least 80%, in some embodiments at least 85%, in some embodiments at least about 90%, in some embodiments at least 91 %, in some embodiments at least 92%, in some embodiments at least 93%, in some embodiments at least 94%, in some embodiments at least 95%, in some embodiments at least 96%, in some embodiments at least 97%, in some embodiments at least 98%, and in some embodiments at least about 99% sequence identity to another nucleic acid and/or amino acid sequence, as determined by the FASTA search method in accordance with Pearson & Lipman, 1988.
  • the percent identity calculation is performed over the full length of the nucleic acid and/or amino acid sequence of
  • a functional equivalent of a nucleotide sequence is a sequence that encodes the same amino acid sequence (i.e., that include one or more functionally equivalent codons).
  • a listing of functionally equivalent codons is presented in Table 1 . Table 1
  • Aspartic Acid Aspartic Acid (Asp or D) GAC; GAU
  • Glycine GGA; GGC; GGG; GGU
  • Histidine Histidine (His or H) CAC; CAU
  • Isoleucine (lie or I) AUA; AUC; AUU
  • Lysine (Lys or K) AAA; AAG
  • Threonine (Thr or T) ACA Threonine (Thr or T) ACA; ACC; ACG; ACU
  • Tyrosine (Tyr or Y) UAC Tyrosine (Tyr or Y) UAC; UAU
  • Arginine (Arg or R) AGA; AGG; CGA; CGC; CGG; CGU
  • a functional equivalent of a given amino acid sequence is an amino acid with one or more conservative amino acid substitutions.
  • Conservative amino acid substitution refers to the substitution of one amino acid for another amino acid of the same class (e.g., valine for glycine, or arginine for lysine).
  • Polypeptides that are functionally equivalent to prouroguanylin and/or prouroguanylin fragments can be made using random mutagenesis on the encoding nucleic acids by techniques well known to those having ordinary skill in the art. It is more likely, however, that such polypeptides will be generated by site-directed mutagenesis (again using techniques well known to those having ordinary skill in the art). These polypeptides can have increased functionality or decreased functionality.
  • Functional equivalents can also include fragments of antibodies that have the same or comparable binding characteristics to those of a whole antibody of the presently disclosed subject matter. Such fragments can contain one or both Fab fragments, the F(ab') 2 fragment, the F(ab') fragment, an Fv fragment, or any other fragment that includes at least one antigen binding domain.
  • the antibody fragments contain all six CDRs of a whole antibody of the presently disclosed subject matter (e.g., comprise CDRs comprising each of SEQ ID NOs: 8-13), although fragments containing fewer than all of such regions, such as three, four, or five CDRs, can also be functional equivalents as defined herein.
  • functional equivalents can be or can combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, and the subclasses thereof, as well as other subclasses as might be appropriate for non- mammalian subjects (e.g., IgY for chickens and other avian species).
  • Functional equivalents also include aptamers and other non-antibody molecules, provided that such molecules have the same or comparable binding characteristics to those of a whole antibody of the presently disclosed subject matter.
  • the antibodies, fragments, and derivatives thereof are selected from the group consisting of monoclonal antibody TAB- 004 produced by hybridoma cell line ATCC No. PTA-1 1550, as well as chimeric antibodies or fragments or derivatives thereof, humanized antibodies or fragments or derivatives thereof, single chain antibodies or fragments or derivatives thereof, Fab fragments thereof, F(ab') 2 fragments thereof, Fv fragments thereof, and Fab' fragments thereof.
  • the antibodies, fragments, and derivatives of the presently disclosed subject matter have the binding characteristics of monoclonal antibody TAB-004.
  • the antibodies, fragments, and derivatives of the presently disclosed subject matter have at least some and in some cases all of the binding characteristics of monoclonal antibody TAB-004. In some embodiments, the antibodies, fragments, and derivatives of the presently disclosed subject matter bind to an epitope present within any of SEQ ID NOs: 1 -3, in some embodiments an epitope present within SEQ ID NO: 3.
  • TAB-004 refers to a mAb that is produced by a hybridoma cell line designated "TAB-004" and that was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 201 10-2209, United States of America, on December 16, 2010 under Accession No. PTA-1 1550 pursuant to the terms of the Budapest Treaty.
  • TAB-004 is a mAb of the IgG isotype that has been found to bind to an epitope present on a human mucin-1 (MUC1 ) polypeptide. More particularly, in some embodiments TAB-004 can bind to an epitope present witin SEQ ID NO: 3.
  • TAB-004 itself comprises the CDR sequences disclosed in SEQ ID NOs. 8-13.
  • MUC1 refers to a molecule defined as follows. MUC1 is one of the epithelial mucin family of molecules. MUC1 is widely expressed on a large number of epithelial cancers and is aberrantly glycosylated making it structurally and antigenically distinct from that expressed by non-malignant cells (see e.g., Barratt-Boyes, 1996; Price et al., 1998; Peterson et al., 1991 ).
  • MUC1 The dominant form of MUC1 is a high molecular weight molecule comprised of a large highly immunogenic extracellular mucin- like domain with a large number of twenty amino acid tandem repeats, a transmembrane region, and a cytoplasmic tail (Quin et al. , 2000; McGucken et al. , 1995; Dong et al., 1997).
  • MUC1 is overexpressed and aberrantly glycosylated in most epithelial adenocarcinomas including breast and pancreas.
  • Adenocarcinoma of the breast and pancreas not only overexpress MUC1 but also shed MUC1 into the circulation.
  • High MUC1 serum levels are associated with progressive disease.
  • MUC1 has been exploited as a prospective biomarker because of the complex and heterogeneous nature of the epitopes expressed within the antigen.
  • MUC1 synthesized by cancerous tissues usually displays an aberrant oligosaccharide profile, which gave rise to the expression of neomarkers such as sialyl-Lea (assayed in the CA19-9 test), sialyl-Lex, and sialyl-Tn (TAG-72), as well as the cryptic epitopes such as Tn.
  • neomarkers such as sialyl-Lea (assayed in the CA19-9 test), sialyl-Lex, and sialyl-Tn (TAG-72)
  • Tn sialyl-Lea
  • TAG-72 sialyl-Tn
  • MUC1 antibody known as PAM4 has gained attention for use in pancreatic cancer diagnosis due to its high sensitivity and specificity for pancreatic cancer but not any other epithelial cancers such as breast and ovarian cancers (Gold et al., 2007).
  • MUC1 is localized to the apical region of the cells. Malignant transformation results in upregulation of MUC1 by gene amplification and/or increased transcriptional activation and the distribution of MUC1 on the cell surface is no longer confined to the apical region (Bieche & Lidereau, 1997). While the function of MUC1 still awaits clarification, high cytoplasmic expression of MUC1 has been associated with poor prognosis in patients with breast and/or ovarian cancers.
  • MUC1 has also been demonstrated to play a role in cell adhesion, cell signaling, and immune responses (Quin et al., 2000; McGucken et al., 1995; Dong et al., 1997; Henderson et al., 1998).
  • a non-limiting example of an amino acid sequence of a MUC1 gene product from humans is presented in SEQ ID NO: 1 .
  • Nucleotide and amino acid sequences of MUC1 gene products from other species include GENBANK® Accession NOs: AAA39755, Q02496, and NP_038633 (mouse), NP_036734 (rat), NP_001 181906 (dog), AA063589 (pig), and NP_776540 (cow).
  • K-ras refers to a K-ras oncogene gene and gene products therefrom (see e.g., Kahn et al., 1987).
  • An exemplary K-ras gene product is a human K-ras gene product including, but not limited to that disclosed as SEQ ID NO: 2, which corresponds to GENBANK® Accession No. NP_004976.
  • K-ras also encompasses mutated forms of K-ras.
  • the terms “mutated K-ras”, “mutant K-ras polypeptide”, and “mutant K-ras protein” are used interchangeably to refer to a K-ras polypeptide comprising at least one K-ras mutation as compared to SEQ ID NO: 2.
  • a mutant K-ras polypeptide comprises a mutation at either amino acid number 12 or 13 of the mature polypeptide (i.e., amino acid position 13 or 14 of SEQ ID NO: 2 since the mature polypeptide would not include the methionine residue at position 1 of SEQ ID NO: 2).
  • a mutant K-ras polypeptide comprises a mutation selected from among a glycine-12 mutation to serine (referred to herein as "G12S"), G12V, G12D, G12A, G12C, G13A, and G13D.
  • G12S glycine-12 mutation to serine
  • G12V glycine-12 mutation to serine
  • G12D G12A
  • G12C G13A
  • G13D G13D
  • SEQ ID NO: 2 A representative example of a mutant K-ras G12D polypeptide to which antibodies of the presently disclosed subject matter bind in part is shown in SEQ ID NO: 2 and described in Kahn et al., 1987.
  • the antibodies, and the fragments and derivatives thereof, of the presently disclosed subject matter bind to a portion of a K-ras polypeptide that comprises a G12D mutation (referred to herein as "mutant K-ras G12D” or “K-ras G12D ").
  • mutant K-ras polypeptides include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, deletion variants, insertion variants, fusion polypeptides, orthologs, and interspecies homologs.
  • a mutant K-ras polypeptide can include one or more additional residues at the C- or N-terminus, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues, and/or fusion protein residues.
  • compositions Comprising Antibodies, Fragments, and/or Derivatives of the Presently Disclosed Subject Matter
  • the presently disclosed subject matter also provides compositions comprising the presently disclosed antibodies, fragments, and/or derivatives.
  • a schematic depiction of exemplary compositions of the presently disclosed subject matter and exemplary uses therefor is provided in Figure 6.
  • a composition of the presently disclosed subject matter comprises the presently disclosed antibodies, fragments, and/or derivatives thereof and one or more pharmaceutically acceptable carriers and/or excipients.
  • the carrier(s) and/or excipient(s) is pharmaceutically acceptable for use in humans.
  • Suitable formulations include aqueous and non-aqueous sterile injection solutions which can contain anti- oxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections
  • Some exemplary ingredients are sodium dodecyl sulfate (SDS) in the range of in some embodiments 0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol or another sugar in the range of in some embodiments 10 to 100 mg/ml, in some embodiments about 30 mg/ml; and/or phosphate-buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used.
  • SDS sodium dodecyl sulfate
  • PBS phosphate-buffered saline
  • compositions of the presently disclosed subject matter can also comprise an active agent, wherein the active agent comprises a therapeutic moiety, a diagnostic moiety, and/or a biologically active moiety.
  • active agent thus refers to a component of the presently disclosed compositions that provides a therapeutic benefit to a subject, permits visualization of cells or tissues in which the compositions of the presently disclosed subject matter accumulate, detection of epitopes to which the presently disclosed antibodies, fragments, and derivatives bind, and/or enhances any of these activities.
  • an active agent of the presently disclosed subject matter is selected from the group consisting of a radioactive molecule (including, but not limited to radionuclides and radioisotopes), a sensitizer molecule, an imaging agent or other detectable agent, a toxin, a cytotoxin, an anti-angiogenic agent, an anti-tumor agent, a chemotherapeutic agent, an immunomodulator, a cytokine, a reporter group, and combinations thereof. It is understood that these categories are not intended to be mutually exclusive, as some radioactive molecules, for example, are also chemotherapeutic agents, some immunomodulators are cytokines, etc.
  • an active agent comprises a chemotherapeutic.
  • chemotherapeutics are known to one of ordinary skill in the art, and include, but are not limited to alkylating agents such as nitrogen mustards (e.g. , Chlorambucil, Cyclophosphamide, Isofamide, Mechlorethamine, Melphalan, Uracil mustard), aziridines (e.g. , Thiotepa), methanesulfonate esters (e.g. , Busulfan), nitroso ureas (e.g. , Carmustine, Lomustine, Streptozocin), platinum complexes (e.g.
  • Cisplatin Cisplatin, Carboplatin
  • bioreductive alkylators e.g. , Mitomycin C, Procarbazine
  • DNA strand breaking agents e.g. , Bleomycin
  • DNA topoisomerase I inhibitors e.g.
  • camptothecin and derivatives thereof including, but not limited to 10- hydroxycamptothecin DNA topoisomerase II inhibitors (e.g., Amsacrine, Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, Mitoxantrone, Etoposide, Teniposide, Podophyllotoxin); DNA minor groove binders (e.g., Plicamycin); anti-metabolites such as folate antagonists (e.g., Methotrexate and trimetrexate), pyrimidine antagonists (e.g., Fluorouracil, Fluorodeoxyuridine, CB3717, Azacytidine, Cytarabine, Floxuridine), purine antagonists (e.g., Mercaptopurine, 6-Thioguanine, Fludarabine, Pentostatin), sugar modified analogs (e.g., Cyctrabine, Fludarabine), and ribonucleotide reductas
  • chemotherapeutics include, but are not limited to Taxol, retinoic acid and derivatives thereof (e.g., 13-cis-retinoic acid, all-trans- retinoic acid, and 9-cis-retinoic acid), sulfathiazole, mitomycin C, mycophenolic acid, sulfadiethoxane, and gemcitabine (4-amino-1 -(2-deoxy- 2,2-difluoro- -D-eryi/7ro-pentofuranosyl)pyhmidin-2(1 H)-on-2',2'-difluoro-2'- deoxycytidine).
  • Taxol retinoic acid and derivatives thereof (e.g., 13-cis-retinoic acid, all-trans- retinoic acid, and 9-cis-retinoic acid)
  • sulfathiazole e.g., 13-cis-retinoic acid, all-trans- reti
  • an active agent comprises an anti-angiogenic agent.
  • anti-angiogenic agents include, but are not limited to inhibitors and/or antagonists of vascular endothelial growth factor (VEGF) family and its receptors (e.g., Bevacizumab and other anti-vascular endothelial growth factor (VEGF) antibodies) and neuropilin-1 antagonists.
  • VEGF vascular endothelial growth factor
  • the compositions of the presently disclosed subject matter can be used with additional adjuvants and/or immunomodulators.
  • immunomodulators include, but are not limited to cytokines (including, but not limited to, the cytokines IFN-a, IFN- ⁇ , IL-2, IL- 4, IL-6, TNF, and other cytokines affecting immune cells), CpG oligodeoxynucleotides (CpG ODN), which function as a dendritic cell activator (Rothenfusser et al., 2002), and the immunomodulators set forth in Table 3.
  • ABH 2(S)-amino-6-boronohexanoic acid
  • BEC S- (2-boronoethyl)-L-cysteine
  • L-NMMA L-N G -monomethyl arginine
  • NCX-4016 nitroaspirin; see Emanueli et al. , 2004; CP-533536: see Cameron et al., 2009; SB-505124: see DeCosta Byfield et al., 2004; SD-505124: see Muller & Scherle, 2006; LY580276: see Sawyer et al.
  • a therapeutically effective amount of a composition of the presently disclosed subject matter is administered to a subject.
  • a "therapeutically effective amount” is an amount of a composition sufficient to produce a measurable biological tumor response (such as, but not limited to an immunostimulatory, an anti-angiogenic response, a cytotoxic response, tumor regression, and/or tumor growth inhibition).
  • a measurable biological tumor response such as, but not limited to an immunostimulatory, an anti-angiogenic response, a cytotoxic response, tumor regression, and/or tumor growth inhibition.
  • Actual dosage levels of active ingredients in a composition of the presently disclosed subject matter can be varied so as to administer an amount of the active agent(s) that is effective to achieve the desired therapeutic response for a particular subject.
  • the selected dosage level will depend upon a variety of factors including the activity of the composition, formulation, the route of administration, combination with other drugs or treatments, tumor size and longevity, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.
  • detectable amount of a composition of the presently disclosed subject matter is administered to a subject.
  • a "detectable amount”, as used herein to refer to a composition refers to a dose of such a composition that the presence of the composition can be determined in vivo or in vitro.
  • a detectable amount will vary according to a variety of factors, including but not limited to chemical features of the composition being labeled, the detectable label, the labeling methods, the method of imaging and parameters related thereto, metabolism of the labeled drug in the subject, the stability of the label (including, but not limited to the half-life of a radionuclide label), the time elapsed following administration of the composition prior to imaging, the route of administration, the physical condition and prior medical history of the subject, and the size and longevity of the tumor or suspected tumor.
  • a detectable amount can vary and can be tailored to a particular application. After study of the present disclosure, it is within the skill of one in the art to determine such a detectable amount.
  • detectable moiety refers to any molecule that can be detected by any moiety that can be added to an antibody, or a fragment or derivative thereof, that allows for the detection of the antibody, fragment, or derivative in vitro and/or in vivo.
  • detectable moieties include, but are not limited to, chromophores, fluorescent moieties, enzymes, antigens, groups with specific reactivity, chemiluminescent moieties, and electrochemically detectable moieties, etc.
  • the antibodies are biotinylated.
  • a detectable moiety comprises a fluorophore.
  • Any fluorophore can be employed with the compositions of the presently disclosed subject matter, provided that the conjugation of fluorophore results in a composition that is detectable either in vivo (e.g., after administration to a subject) and/or in vitro, and further does not negatively impact the ability of the antibody, or the fragment or derivative thereof, to bind to its epitope.
  • Representative fluorophores include, but are not limited to 7- dimethylaminocoumarin-3-carboxylic acid, dansyl chloride, nitrobenzodiazolamine (NBD), dabsyl chloride, cinnamic acid, fluorescein carboxylic acid, Nile Blue, tetramethylcarboxyrhodamine, tetraethylsulfohodamine, 5-carboxy-X-rhodamine (5-ROX), and 6-carboxy-X- rhodamine (6-ROX). It is understood that these representative fluorophores are exemplary only, and additional fluorophores can also be employed.
  • ALEXA FLUOR® dye series includes at least 19 different dyes that are characterized by different emission spectra. These dyes include ALEXA FLUOR® 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750 (available from Invitrogen Corp., Carlsbad, California, United States of America), and the choice of which dye to employ can be made by the skilled artisan after consideration of the instant specification based on criteria including, but not limited to the chemical compositions of the specific ALEXA FLUOR®, whether multiple detectable moieties are to be employed and the emission spectra of each, the detection technique to be employed, etc.
  • a detectable moiety comprises a cyanine dye.
  • Non-limiting examples of cyanine dyes that can be conjugated to the antibodies, fragments, and/or derivatives of the presently disclosed subject matter include the succinimide esters Cy5, Cy5.5, and Cy7, supplied by Amersham Biosciences (Piscataway, New Jersey, United States of America).
  • a detectable moiety comprises a near infrared (NIR) dye.
  • NIR near infrared
  • the biotinylated antibodies are detected using a secondary antibody that comprises an avidin or streptavidin group and is also conjugated to a fluorescent label including, but not limited to Cy3, Cy5, Cy7, and any of the ALEXA FLUOR®® series of fluorescent labels available from INVITROGENTM (Carlsbad, California, United States of America).
  • the antibody, fragment, or derivative thereof is directly labeled with a fluorescent label and cells that bind to the antibody are separated by fluorescence-activated cell sorting. Additional detection strategies are known to the skilled artisan.
  • the antibodies of the presently disclosed subject matter can be labeled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • a detectable moiety can be a radioisotope, such as but not limited to 3 H, 14 C, 32 P, 35 S, 125 l, or 3 l; a fluorescent or chemiluminescent compound such as but not limited to fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as but not limited to alkaline phosphatase, ⁇ -galactosidase, or horseradish peroxidase.
  • a targeting ligand of the presently disclosed subject matter comprises a detectable label such as a fluorescent label, an epitope tag, or a radioactive label, each described briefly herein below.
  • Fluorescence Any detectable fluorescent dye can be used, including but not limited to FITC (fluorescein isothiocyanate), FLUOR XTM, ALEXA FLUOR®, OREGON GREEN®, TMR (tetramethylrhodamine), ROX (X- rhodamine), TEXAS RED®, BODIPY® 630/650, and Cy5 (available from Amersham Pharmacia Biotech of Piscataway, New Jersey, United States of America, or from Molecular Probes Inc. of Eugene, Oregon, United States of America).
  • FITC fluorescein isothiocyanate
  • FLUOR XTM fluorescein isothiocyanate
  • ALEXA FLUOR® OREGON GREEN®
  • TMR tetramethylrhodamine
  • ROX X- rhodamine
  • TEXAS RED® BODIPY® 630/650
  • Cy5 available from Amersham Pharmacia Biotech of
  • a fluorescent label can be detected directly using emission and absorbance spectra that are appropriate for the particular label used.
  • Common research equipment has been developed for in vitro detection of fluorescence, including instruments available from GSI Lumonics (Watertown, Massachusetts, United States of America) and Genetic Microsystems Inc. (Woburn, Massachusetts, United States of America). Most of the commercial systems use some form of scanning technology with photomultiplier tube detection. Criteria for consideration when analyzing fluorescent samples are summarized by Alexay et al., 1996.
  • an epitope label a protein or compound that binds the epitope can be used to detect the epitope.
  • a representative epitope label is biotin, which can be detected by binding of an avidin-conjugated fluorophore, for example avidin-FITC.
  • the label can be detected by binding of an avidin-horseradish peroxidase (HRP) streptavidin conjugate, followed by colorimetric detection of an HRP enzymatic product.
  • HRP avidin-horseradish peroxidase
  • the production of a colorimetric or luminescent product/conjugate is measurable using a spectrophotometer or luminometer, respectively.
  • Autoradiographic Detection In the case of a radioactive label (e.g., 131 1 or 99m Tc) detection can be accomplished by conventional autoradiography or by using a phosphorimager as is known to one of skill in the art.
  • a preferred autoradiographic method employs photostimulable luminescence imaging plates (Fuji Medical Systems of Stamford, Connecticut, United States of America). Briefly, photostimulable luminescence is the quantity of light emitted from irradiated phosphorous plates following stimulation with a laser during scanning. The luminescent response of the plates is linearly proportional to the activity (Amemiya et al., 1988; Hallahan et al. , 2001 a).
  • compositions of the presently disclosed subject matter can further comprise a drug carrier to facilitate drug preparation and administration.
  • a drug delivery vehicle or carrier can be used, including but not limited to a gene therapy vector (e.g., a viral vector or a plasmid), a microcapsule, for example a microsphere or a nanosphere (Manome et al. , 1994; Hallahan et al., 2001 b; Saltzman & Fung, 1997), a peptide (U.S. Patent Nos. 6,127,339 and 5,574,172), a glycosaminoglycan (U.S. Patent No. 6,106,866), a fatty acid (U.S. Patent No.
  • Antibodies, fragments, or derivatives can also be coupled to drugs or drug carriers using methods known in the art, including but not limited to carbodiimide conjugation, esterification, sodium periodate oxidation followed by reductive alkylation, and glutaraldehyde crosslinking (see e.g., Goldman et al., 1997; Cheng, 1996; Neri et al., 1997; Nabel, 1997; Park et al., 1997; Pasqualini et al., 1997; Bauminger & Wilchek, 1980; U.S. Patent No. 6,071 ,890; and European Patent No. 0 439 095.
  • Suitable methods for administration of a composition of the presently disclosed subject matter include, but are not limited to intravascular, subcutaneous, intramuscular, and intratumoral administration.
  • intravascular administration is employed.
  • the phrases "intravascular administration” and “intravascular provision” refer to administration of a composition directly into the vascular network of a subject.
  • Techniques that can be employed for intravascular administration of compositions are known to those of skill in the art, and include, but are not limited to intravenous administration and intraarterial administration. It is understood that any site and method for intravascular administration can be chosen, depending at least in part on the species of the subject to which the composition is to be administered.
  • compositions can be administered as an aerosol or coarse spray.
  • the antibodies, and/or the fragments and/or derivatives thereof, of the presently disclosed subject matter also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety such as a radio-opaque agent and/or a radioisotope is administered to a subject, in some embodiments via intravenous administration, and the presence and location of the labeled antibody in the host is assayed.
  • a detectable moiety such as a radio-opaque agent and/or a radioisotope
  • a composition of the presently disclosed subject matter comprises a label that can be detected in vivo.
  • in vivo refers to generally non-invasive methods such as scintigraphic methods, magnetic resonance imaging, ultrasound, or fluorescence, each described briefly herein below.
  • non-invasive methods does not exclude methods employing administration of a contrast agent to facilitate in vivo imaging.
  • the detectable moiety can be conjugated or otherwise associated with an antibody, fragment, or derivative of the presently disclosed subject matter, a therapeutic, a diagnostic agent, a drug carrier, or combinations thereof as set forth in more detail hereinabove.
  • time sufficient for binding refers to a temporal duration that permits binding of the labeled agent to a radiation-induced target molecule.
  • Scintigraphic Imaging Scintigraphic imaging methods include SPECT (Single Photon Emission Computed Tomography), PET (Positron Emission Tomography), gamma camera imaging, and rectilinear scanning.
  • a gamma camera and a rectilinear scanner each represent instruments that detect radioactivity in a single plane.
  • Most SPECT systems are based on the use of one or more gamma cameras that are rotated about the subject of analysis, and thus integrate radioactivity in more than one dimension.
  • PET systems comprise an array of detectors in a ring that also detect radioactivity in multiple dimensions.
  • Imaging instruments suitable for practicing the detection and/or imaging methods of the presently disclosed subject matter, and instruction for using the same, are readily available from commercial sources. Both PET and SPECT systems are offered by ADAC of Milpitas, California, United States of America, and Siemens of Hoffman Estates, Illinois, United States of America. Related devices for scintigraphic imaging can also be used, such as a radio- imaging device that includes a plurality of sensors with collimating structures having a common source focus.
  • the detectable label comprises in some embodiments a radionuclide label, in some embodiments a radionuclide label selected from the group consisting of 18 F, 64 Cu, 65 Cu, 67 Ga, 68 Ga, 77 Br, 80m Br, 95 Ru, 97 Ru, 103 Ru, 105 Ru, 99m Tc, 107 Hg, 203 Hg, 123 l, 124 l, 125 l, 126 l, 131 l, 133 l, 111 ln, 113m ln, 99m Re, 105 Re, 101 Re, 186 Re, 188 Re, 121m Te, 122m Te, 125m Te, 165 Tm, 167 Tm, 168 Tm, and nitride or oxide forms derived there from.
  • the radionuclide label comprises 131 1 or 99m Tc.
  • a targeting molecule can be derivatized so that a radioisotope can be bound directly to it (Yoo et al., 1997).
  • a linker can be added that to enable conjugation.
  • Representative linkers include diethylenetriamine pentaacetate (DTPA)-isothiocyanate, succinimidyl 6-hydrazinium nicotinate hydrochloride (SHNH), and hexamethylpropylene amine oxime (HMPAO; Chattopadhyay et al., 2001 ; Sagiuchi et al., 2001 ; and U.S. Patent No. 6,024,938). Additional methods can be found in U.S. Patent No. 6,080,384; Hnatowich et al., 1996; and Tavitian et al., 1998.
  • the labeling moiety is a radionuclide
  • stabilizers to prevent or minimize radiolytic damage such as ascorbic acid, gentisic acid, or other appropriate antioxidants, can be added to the composition comprising the labeled targeting molecule.
  • Magnetic Resonance Imaging Magnetic Resonance Imaging (MRI). Magnetic resonance image- based techniques create images based on the relative relaxation rates of water protons in unique chemical environments.
  • magnetic resonance imaging refers to magnetic source techniques including convention magnetic resonance imaging, magnetization transfer imaging (MTI), proton magnetic resonance spectroscopy (MRS), diffusion-weighted imaging (DWI) and functional MR imaging (fMRI; see e.g., Rovaris et al., 2001 ; Pomper & Port, 2000; and references cited therein).
  • MMI magnetization transfer imaging
  • MRS proton magnetic resonance spectroscopy
  • DWI diffusion-weighted imaging
  • fMRI functional MR imaging
  • Contrast agents for magnetic source imaging include but are not limited to paramagnetic or superparamagnetic ions, iron oxide particles (Weissleder et al., 1992; Shen et al., 1993), and water-soluble contrast agents.
  • Paramagnetic and superparamagnetic ions can be selected from the group of metals including iron, copper, manganese, chromium, erbium, europium, dysprosium, holmium and gadolinium.
  • Preferred metals are iron, manganese and gadolinium; most preferred is gadolinium.
  • metal ions can be bound by chelating moieties, which in turn can be conjugated to a therapeutic agent in accordance with the methods of the presently disclosed subject matter.
  • gadolinium ions are chelated by diethylenetriaminepentaacetic acid (DTPA).
  • Lanthanide ions are chelated by tetraazacyclododocane compounds. See U.S. Patent Nos. 5,738,837 and 5,707,605.
  • a contrast agent can be carried in a liposome (Schwendener, 1992).
  • Images derived used a magnetic source can be acquired using, for example, a superconducting quantum interference device magnetometer (SQUID, available with instruction from Quantum Design of San Diego, California, United States of America; see also U.S. Patent No. 5,738,837).
  • SQUID superconducting quantum interference device magnetometer
  • Ultrasound imaging can be used to obtain quantitative and structural information of a target tissue, including a tumor.
  • Administration of a contrast agent can enhance visualization of the target tissue during an ultrasound examination.
  • the contrast agent can be selectively targeted to the target tissue of interest, for example by using a peptide for guided drug delivery (e.g., radiation guided drug delivery) as disclosed herein.
  • Representative agents for providing microbubbles in vivo include but are not limited to gas-filled lipophilic or lipid— based bubbles (e.g. , U.S. Patent Nos. 6,245,318; 6,231 ,834; 6,221 ,018; and 5,088,499).
  • gas or liquid can be entrapped in porous inorganic particles that facilitate microbubble release upon delivery to a subject (U.S. Patent Nos. 6,254,852 and 5,147,631 ).
  • Gases, liquids, and combinations thereof suitable for use with the presently disclosed subject matter include air; nitrogen; oxygen; is carbon dioxide; hydrogen; nitrous oxide; an inert gas such as helium, argon, xenon or krypton; a sulfur fluoride such as sulfur hexafluoride, disulfur decafluoride or trifluoromethylsulfur pentafluoride; selenium hexafluoride; an optionally halogenated silane such as tetramethylsilane; a low molecular weight hydrocarbon (e.g.
  • alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclobutane or cyclopentane, an alkene such as propene or a butene, or an alkyne such as acetylene; an ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms); or a mixture of any of the foregoing.
  • Halogenated hydrocarbon gases can show extended longevity, and thus are preferred for some applications.
  • gases of this group include decafluorobutane, octafluorocyclobutane, decafluoroisobutane, octafluoropropane, octafluorocyclopropane, dodecafluoropentane, decafluorocyclopentane, decafluoroisopentane, perfluoropexane, perfluorocyclohexane, perfluoroisohexane, sulfur hexafluoride, and perfluorooctaines, perfluorononanes; perfluorodecanes, optionally brominated.
  • Attachment of targeting ligands to lipophilic bubbles can be accomplished via chemical crosslinking agents in accordance with standard protein-polymer or protein-lipid attachment methods (e.g. , via carbodiimide (EDC) or thiopropionate (SPDP)).
  • EDC carbodiimide
  • SPDP thiopropionate
  • large gas- filled bubbles can be coupled to a targeting ligand using a flexible spacer arm, such as a branched or linear synthetic polymer (U.S. Patent No. 6,245,318).
  • a targeting ligand can be attached to the porous inorganic particles by coating, adsorbing, layering, or reacting the outside surface of the particle with the targeting ligand (U.S. Patent No. 6,254,852).
  • Non-invasive imaging methods can also comprise detection of a fluorescent label.
  • a drug comprising a lipophilic component can be labeled with any one of a variety of lipophilic dyes that are suitable for in vivo imaging (see e.g., Heredia et al., 1991 ; Ragnarson et al., 1992; Fraser, 1996).
  • Representative labels include but are not limited to carbocyanine and aminostyryl dyes, preferably long chain dialkyl carbocyanines (e.g., Dil, DiO, and DiD available from Molecular Probes Inc. of Eugene, Oregon, United States of America) and dialkylaminostyryl dyes.
  • Lipophilic fluorescent labels can be incorporated using methods known to one of skill in the art. For example VYBRANTTM cell labeling solutions are effective for labeling of cultured cells of other lipophilic components (Molecular Probes Inc. of Eugene, Oregon, United States of America).
  • a fluorescent label can also comprise sulfonated cyanine dyes, including Cy5.5 and Cy5 (available from Amersham of Arlington Heights, Illinois, United States of America), IRD41 and IRD700 (available from Li-Cor, Inc. of Lincoln, Iowa), NIR-1 (available from Dejindo of Kumamoto, Japan), and LaJolla Blue (available from Diatron of Miami, Florida, United States of America; see also Licha et al., 2000; Weissleder et al., 1999; Vinogradov et al. , 1996).
  • Cy5.5 and Cy5 available from Amersham of Arlington Heights, Illinois, United States of America
  • IRD41 and IRD700 available from Li-Cor, Inc. of Lincoln, Iowa
  • NIR-1 available from Dejindo of Kumamoto, Japan
  • LaJolla Blue available from Diatron of Miami, Florida, United States of America; see also Licha et al., 2000; Weissleder et al., 1999; Vinogradov
  • a fluorescent label can comprise an organic chelate derived from lanthanide ions, for example fluorescent chelates of terbium and europium (U.S. Patent No. 5,928,627).
  • Such labels can be conjugated or covalently linked to a drug as disclosed therein.
  • an image is created using emission and absorbance spectra that are appropriate for the particular label used.
  • the image can be visualized, for example, by diffuse optical spectroscopy. Additional methods and imaging systems are described in U.S. Patent Nos. 5,865,754; 6,083,486; and 6,246,901 , among other places.
  • the antibodies, fragments, and/or derivatives of the presently disclosed subject matter are employed for in vivo imaging of tumors, wherein a composition of the presently disclosed subject matter that has been labeled with an imaging moiety such as a radio-opaque agent, a radioisotope, or other imaging agent is administered to a subject, and the presence and location of the detectibly-labeled composition in the subject is assayed.
  • an imaging moiety such as a radio-opaque agent, a radioisotope, or other imaging agent
  • an antibody is labeled with any moiety that is detectable in situ in a subject, for example by nuclear magnetic resonance, radiology, or other detection methods known in the art.
  • the presently disclosed subject matter also provides methods for detecting tumors in subjects.
  • the presently disclosed methods comprise (a) administering to the subject a composition comprising the antibody, or the fragment or derivative thereof, of the presently disclosed subject matter conjugated to a detectable label; and (b) detecting the detectable label to thereby detect the tumor.
  • the tumor is a tumor of the pancreas, breast, ovary, colon, or rectum, and/or a metastatic cell derived therefrom, which optionally expresses MUC1 , a mutant K-ras, or both.
  • the detectable label comprises an imaging agent selected from the group consisting of paramagnetic, radioactive, and fluorogenic ions including, but not limited to those set forth in more detail hereinabove.
  • the radioactive imaging agent can be, for example, gamma- emitters, positron-emitters, x-ray-emitters, or any other agents for which a detection method is available.
  • radioactive imaging agents include 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 77 Br, 81 Rb/ 81 MKr, 87M Sr, 99M Tc, 111 ln, 113 ln, 123 l, 125 l, 127 Cs, 129 Cs, 131 l, 132 l, 197 Hg, 203 Pb, and 206 Bi, but the presently disclosed subject matter is not limited to just these radioisotopes. The presently disclosed subject matter also provides methods for treating tumors.
  • the methods comprise administering to the subject a composition comprising an antibody, or a fragment or derivative thereof of the presently disclosed subject matter conjugated to an active agent, whereby the active agent contacts the tumor to thereby treat the tumor.
  • active agents are disclosed herein, and include but are not limited to therapeutic agents, optionally chemotherapeutic agents, toxins, radiotherapeutic agents, and combinations of any of the foregoing.
  • a composition of the presently disclosed subject matter can comprise an antibody, or a fragment or derivative thereof as disclosed herein conjugated to a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from among an anti-tumor drug, a cytokine, an anti-metabolite, an alkylating agent, a hormone, methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, 5-fluorouracil, melphalan, chlorambucil, a nitrogen mustard, cyclophosphamide, cis- platinum, vindesine, vinca alkaloids, mitomycin, bleomycin, purothionin, macromomycin, 1 ,4-benzoquinone derivatives, trenimon, steroids, aminopterin, anthracyclines, demecolcine, etoposide, mithramycin, doxorubicin, daunomycin, vinblast
  • composition of the presently disclosed subject matter can comprise an antibody, or a fragment or derivative thereof as disclosed herein conjugated to a toxin.
  • exemplary toxins include, but are not limited to Russell's Viper Venom, activated Factor IX, activated Factor X, thrombin, phospholipase C, cobra venom factor, ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, gelonin, saporin, modeccin, viscumin, volkensin, and combinations thereof.
  • compositions of the presently disclosed subject matter can also comprise an antibody, or a fragment or derivative thereof as disclosed herein conjugated to a radiotherapeutic agent.
  • radiotherapeutic agents include, but are not limited to 47 Sc, 67 Cu, 90 Y, 109 Pd, 123 l, 125 l, 131 l, 186 Re, 188 Re, 199 Au, 211 At, 212 Pb, 212 Bi, 32 P, 33 P, 71 Ge, 77 As, 103 Pb, 105 Rh, 111 Ag, 119 Sb, 121 Sn,
  • the presently disclosed subject matter also provides methods for suppressing tumor growth in a subject.
  • the methods comprise administering to a subject bearing a tumor an effective amount of an isolated antibody, fragment, or derivative of the presently disclosed subject matter.
  • the antibody, fragment, or derivative of the presently disclosed subject matter binds to an epitope present within any of SEQ ID NOs: 1 -3.
  • the tumor is a tumor of the pancreas, breast, ovary, colon, or rectum, and/or a metastatic cell derived therefrom, which in some embodiments expresses MUC1 , a mutant K-ras, or both.
  • compositions and methods disclosed herein as part of a combination therapy.
  • the presently disclosed subject matter provides in some embodiments administering to the subject one or more additional anti-tumor treatments.
  • additional anti-tumor treatments include but are not limited to radiotherapy, chemotherapy, an additional immunotherapy, an antiinflammatory therapy, and combinations thereof.
  • an anti-inflammatory therapy can comprise administering to the subject an anti-inflammatory agent such as, but not limited to a nonsteroidal anti-inflammatory drug (NSAID).
  • NSAIDs include, but are not limited to cyclooxygenase inhibitors (e.g., indomethacin), particularly cyclooxygenase-2-specific inhibitors such as, but not limited to celecoxib and rofecoxib.
  • Combination therapies can also include administration of one or more additional anti-tumor therapies such as, but not limited to administering gemcitabine, which is 4-amino-1 -(2-deoxy-2,2-difluoro- -D-erythro- pentofuranosyl)pyrimidin-2(1 H)-on-2',2'-difluoro-2'-deoxycytidine; celecoxib, which is 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1 - yljbenzenesulfonamide, pharmaceutically acceptable salts thereof, and/or combinations thereof to the subject.
  • Combination therapies can also include administration of ionizing radiation to the subject, before, during, and/or after the administration course of any of the compositions of the presently disclosed subject matter.
  • the antibodies, fragments, derivatives, and/or conjugates thereof can be administered to a subject, for example in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the antibodies and/or conjugates can also be administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects, as desired.
  • Suitable pharmaceutically acceptable carriers, diluents, and/or excipients are well known and can be employed by those of skill in the art as the clinical situation warrants.
  • suitable carriers, diluents, and/or excipients include: (1 ) Dulbecco's phosphate buffered saline, pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCI), and (3) 5% (w/v) dextrose.
  • the antibody When present in an aqueous dosage form, rather than being lyophilized, the antibody typically will be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml, although wide variation outside of these ranges is permitted.
  • compositions and methods of the presently disclosed subject matter can be employed in vitro, in vivo, or ex vivo.
  • compositions and methods of the presently disclosed subject matter can be used for screening and/or treatment of a cancer in which MUC1 or mutated K-ras expression is elevated.
  • cancers include, but are not limited to, cancers of the ovary, breast, lung, pancreas, and prostate.
  • an appropriate dosage of an antibody, fragment, or derivative thereof, and/or a conjugate thereof of the presently disclosed subject matter can depend on the type of disease to be treated, the severity and course of the disease, whether the antibodies and/or conjugates are administered for preventive or therapeutic purposes, the course of previous therapy, the patient's clinical history and response to the antibodies and/or conjugates, and the discretion of the attending physician.
  • the antibodies and/or conjugates of the presently disclosed subject matter can be administered to a subject at one time or over a course of several or many treatments.
  • the presently disclosed subject matter also provides methods for detecting, purifying, and targeting tumor cells, cancer stem cells, or both present in a subject or isolated from a subject using the antibodies, or the fragments or derivatives thereof, disclosed herein.
  • the presently disclosed subject matter provides methods for detecting tumor cells, cancer stem cells, or both by detecting the binding of an antibody, or a fragment or derivative thereof to tumor cells, cancer stem cells, or both present in biological samples isolated from subjects who had and/or presently have a cancer.
  • the compositions disclosed herein that employ detectable labels can be employed for this purpose.
  • the presently disclosed subject matter provides methods for purifying cancer stem cells.
  • the methods comprise (a) providing a population of cells suspected of comprising cancer stem cells; (b) identifying a subpopulation of the cells that bind to an antibody, or a fragment or derivative thereof, the binds to an epitope present within any of SEQ ID NOs: 1 -3; and (b) purifying the subpopulation.
  • the population of cells comprises circulating cells isolated from a subject that has a cancer.
  • the methods further comprise removing lineage positive (lin + ) cells from the population of cells before the identifying step or removing lin + cells from the purified subpopulation.
  • removing lin + cells from cell populations are known in the art. An exemplary method is as follows:
  • Single cell suspensions are either isolated from a subject (e.g., from blood, lymph fluids, bone marrow aspirates, etc.) or are prepared from tissues.
  • tissues sections from a tissue suspected of having cancer stem cells (e.g., pancreatic adenocarcinoma tissue) can be mechanically homogenized and digested with collagenase IV and DNase for 30 minutes at 37°C.
  • Lineage cell depletion using, for example, one of the several species-specific Lineage Cell Depletion Kits sold by Miltenyi Biotec (Bergisch Gladbach, Germany), which remove cells expressing the following lineage antigens: CD2, CD3, CD1 1 b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a from the cell suspensions.
  • the lineage negative subpopulation can then be then screened using flow cytometry for cells expressing MUC1 using, for example, the monoclonal TAB-004 antibody of the presently disclosed subject matter. It is understood that the steps of the various selections can be performed in any order. If desired, antibodies directed against the stem cell markers CD133 (AC133) and/or CD24 + /CD44 + can also be employed.
  • the presently disclosed subject matter also provides methods for targeting an active agent to a circulating cancer stem cell in a subject.
  • the methods comprise contacting a cancer stem cell (optionally a circulating cancer stem cell) with a composition comprising an antibody, or a fragment or derivative thereof, of the presently disclosed subject matter and an active agent.
  • the composition thus delivers the active agent to the cancer stem cell.
  • the active agent comprises a therapeutic agent, a chemotherapeutic agent, a toxin, a radiotherapeutic agent, or a combination thereof.
  • the therapeutic agent comprises an immunomodulator, which in some embodiments could one or more of an indoleamine 2,3-dioxygenase (IDO) inhibitor (e.g., 1 -methyl-DL-tryptophan (1 MT)); an EP2/EP4 receptor antagonist; a CXCR4 antagonist, a vascular endothelial growth factor receptor 1 antagonist, Celebrex, a TGF RI antagonist, and a dendritic cell activator.
  • IDO indoleamine 2,3-dioxygenase
  • breast cancer and pancreatic cancer are the third and fourth leading cause of cancer related deaths, respectively.
  • Breast cancer is the most commonly diagnosed cancer among women, but while pancreatic cancer is less common, it has worst prognosis of all cancers.
  • the poor prognosis associated with pancreatic cancer results at least in part from a lack of early detection methods resulting in diagnosis of the disease at an advanced stage.
  • mammography has significantly improved early detection in breast cancer, it is not without its shortcomings. Up to 20% of breast cancers are missed, and false-positive results can lead to anxiety and expensive additional testing.
  • the lack of specificity in mammography screening can lead to the "over-diagnosis" of benign tumors and unnecessary treatment.
  • CT computed tomography
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • the presently disclosed subject matter also provides methods for predicting the recurrence of cancer in a subject.
  • the methods comprise (a) isolating a biological sample comprising circulating cells from a subject with a cancer; (b) contacting the biological sample with one or more of the antibodies, fragments, or derivatives of the presently disclosed subject matter; and (c) identifying in the biological sample one or more circulating cells that bind to the one or more of the antibodies, fragments, or derivatives of the presently disclosed subject matter, whereby the recurrence of a cancer is predicted in the subject.
  • the identification of circulating cells that bind to the antibodies, fragments, and/or derivatives of the presently disclosed subject matter can be indicative of a recurrence of a subject's cancer when the subject had previously been negative for such circulating cells.
  • the presence of circulating cells that bind to the one or more of the antibodies, fragments, or derivatives of the presently disclosed subject matter indicates that the subject is at enhanced risk of metastatic disease relative to a subject that is negative for such circulating cells.
  • the presently disclosed subject matter also provides methods for prognosing progression of a cancer in subjects.
  • the methods comprise isolating a biological sample comprising circulating cells from a subject with a cancer; contacting the biological sample with the antibody, or the fragment or derivative thereof, of the presently disclosed subject matter under conditions sufficient for the antibody, or the fragment or derivative thereof, to bind to an epitope present on a tumor and/or a cancer cell, if present, in the biological sample; and identifying in the biological sample one or more circulating cells that bind to the antibody, or the fragment or derivative thereof, whereby progression of a cancer is prognosed in the subject.
  • the biological sample comprises a blood sample, a lymph sample, or a fraction thereof.
  • the cancer is a pancreatic cancer or a breast cancer.
  • the antibody is a monoclonal antibody produced by hybridoma cell line TAB-004 deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 201 10- 2209, United States of America, on December 16, 2010 under the terms of the Budapest Treaty as Accession No. PTA-1 1550.
  • ATCC American Type Culture Collection
  • the fragment or derivative thereof is selected from the group consisting of a chimeric antibody, or a fragment or derivative thereof; a humanized antibody, or a fragment or derivative thereof; a human antibody, or a fragment or derivative thereof; a single chain antibody, or a fragment or derivative thereof; and a Fab fragment, wherein the chimeric antibody, the humanized antibody, the human antibody, the single chain antibody, or the Fab fragment comprises the CDRs of monoclonal antibody TAB-004, and further wherein the chimeric antibody, the humanized antibody, the human antibody, the single chain antibody, or the Fab fragment comprises the CDRs of monoclonal antibody TAB-004.
  • the CDRs of monoclonal antibody TAB-004 comprise heavy chain CDR1 comprising SEQ ID NO: 8; heavy chain CDR2 comprising SEQ ID NO: 9; heavy chain CDR3 comprising SEQ ID NO: 10; light chain CDR1 comprising SEQ ID NO: 1 1 ; light chain CDR2 comprising SEQ ID NO: 12; and light chain CDR3 comprising SEQ ID NO: 13.
  • progression of a cancer refers to evaluating indicia of a cancer disease at a given time point and comparing the same to the indicia of the cancer disease taken at an earlier time point, wherein the comparison is indicative of a progression of the cancer in the subject.
  • progression of the cancer comprises metastasis of the cancer in the subject.
  • the antibody, or the fragment or derivative thereof, of the presently disclosed subject matter can be employed to detect the presence of circulating tumor cells (CTCs) in the blood or other biological samples of cancer patients, as the presence of CTCs can be an important indicator of the potential for metastatic disease and poor prognosis.
  • CTCs circulating tumor cells
  • the CELLSEARCH® system (Veridex, LLC, Raritan, New Jersey, United States of America) is the only method approaved by the United States Food and Drug Administration (FDA) to measure CTCs in metastatic breast, colorectal, and prostate cancer patients.
  • FDA United States Food and Drug Administration
  • EpCAM-expressing CTCs are currently minimally predictive of metastasis.
  • low EpCAM expression by micrometastases has been reported, and attempts to isolate CTCs using antibodies against EpCAM have not been successful to date.
  • MUC1 -expressing cells have high metastatic potential and MUC1 has been found to be expressed on CTCs
  • the presently disclosed antibodies, and the fragments and derivatives thereof, including but not limited to the TAB-004 antibody can serve as a highly reliable and improved predictor of metastasis compared to strategies based on attempting to detect EpCAM-expressing CTCs.
  • the antibodies of the presently disclosed subject matter can also be employed in various assay methods, such as but not limited to competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see e.g., Zola, 1987; Harlow & Lane, 1988).
  • the antibodies of the presently disclosed subject matter also are useful as affinity purification agents.
  • one or more antibodies are immobilized on a suitable support (such as, but not limited to a Sephadex resin or filter paper) using methods well known in the art. See e.g., Harlow & Lane, 1988.
  • Pancreatic Adenocarcinoma Mice Expressing Human MUC1 A strategy for generating a triple transgenic mouse line that expressed human MUC1 and develops pancreatic adenocarcinoma is depicted in Figure 3. Briefly, P48 Cre/+ mice, which expressed the Cre recombinase throughout the developing and adult pancreas (Kawaguchi et al., 2002) were bred to LSL- Kras G12D/+ mice, which contained a transcriptionally inactive K-ras G12D allele that was activated in cells expressing Cre (Jackson et al., 2001 ; Kawaguchi et al. , 2002).
  • mice The progeny that were positive for both P48 Cre/+ and LSL-Kras G12D/+ (designated "PDA mice") were mated to a transgenic mouse line (MUCl .Tg) that carried a human MUC1 transgene and were maintained as heterozygotes (see Figure 3).
  • MUCl .Tg mice expressed human MUC1 , exhibited B- and T- cell compartment tolerance, and were refractory to immunization with the protein encoded by the transgene (Rowse et al., 1998). Since the human MUC1 transgene was driven by its own promoter in these mice, its expression levels were tissue-specific and appropriate. Low-level luminal surfaces of simple epithelial tissue and increased expression in tumors were observed.
  • mice that were positive for P48 Cre/+ , LSL-Kras G12D/+ , and human MUC1 carried three transgenes. All PDA x MUCl .Tg mice developed pancreatic intraepithelial neoplasia (PanlNs) of different stages including PanlN-IA, PanlN-IB, PanlN-2, PanlN-3, and adenocarcinoma (Tinder et al., 2008; Mukherjee et al., 2009). Representative sections from various ages of the PDA x MUCl .Tg pancreas are shown in Figure 1 B. Approximately 80% of these mice developed adenocarcinoma by 26 weeks of age, and almost 100% of the mice developed adenocarcinoma by 34 weeks of age.
  • TAB-004 monoclonal antibody (mAb) described herein bound to an epitope present within SEQ ID NO: 3.
  • the antibody reacted strongly with tumor tissue isolated from human pancreas ( Figure 1 B), human breast ( Figured 2A and 2B), but did not bind appreciably to normal pancreas or breast tissue (see Figures 1 A and 2C).
  • TAB-004 antibody cross reacted with mutated K-ras such that tumor tissues expressing the K-ras mutation but not human MUC1 also showed positive staining with the antibody. All metastatic lesions showed positive reactivity, and it appeared that TAB-004 could bind to an epitope present within a mutated K-ras polypeptide.
  • the TAB-004 antibody was tested with various samples to determine its ability to bind to and sort tumor cells that were present in different environments and under different conditions using Fluorescence Activating Cell Sorting (FACS).
  • FACS Fluorescence Activating Cell Sorting
  • the TAB-004 antibody was employed for staining cells from purified populations of CD133 + and CD24 + /CD44 + /EpCAM + cells.
  • Sections from pancreatic adenocarcinomas are mechanically homogenized and digested in collagenase IV and DNase for 30 minutes at 37°C.
  • Whole blood and single cell suspension from the tumor were subjected to lineage cell depletion using the Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, Catalogue No.
  • Lineage negative (lin " ) cells from blood samples or tumor samples from patients with pancreatic cancer were then screened using flow cytometry for cells expressing MUC1 , using the TAB-004 antibody, and the following pancreatic stem markers CD133 (AC133) or CD24 + /CD44 + .
  • Figures 7A and 7B are histograms of fluorescence-activated cell sorting (FACS) separations of CD133 + ( Figure 7A) and CD247CD447EpCAM + ( Figure 7B) cells and the extent to which the TAB-004 antibody disclosed herein bound to these populations.
  • FACS fluorescence-activated cell sorting
  • FIG 8 the distributions of cells in histologically normal pancreatic tissue (Figure 8C) versus adjacent pancreatic adenocarcinoma tissue ( Figure 8D) stained with the TAB-004 antibody versus a CXCR4 antibody was compared. As can be seen, cells that were positive for MUC1 or for CXCR4 were more abundant in pancreatic adenocarcinoma tissue than in histologically normal adjacent pancreatic tissue.
  • Figures 8A and 8B show the results of negative controls ( Figure 8A - no antibody; Figure 8B - isotype control antibody).
  • FIG. 9 provides a series of FACS plots that show that the TAB-004 antibody was superior to a standard EpCAM antibody in detecting circulating tumor cells in pancreatic cancer patients.
  • the TAB-004 antibody of the presently disclosed subject matter was conjugated to 1 -methyl-DL-tryptophan (1 MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor; an EP2/EP4 receptor antagonist; and CpG oligodeoxynucleotides (CpG ODN), which function as dendritic cell activators (Rothenfusser et a/. , 2002).
  • CpG ODN CpG oligodeoxynucleotides
  • Data on the functional role of the TAB-004 antibody conjugated to CpG ODN is provided herewith as a non-limiting example of the functionality of the antibodies and conjugates of the presently disclosed subject matter.
  • the TAB-004 antibody alone or conjugated to CpG ODN bound to a tumor cell line (referred to herein as the "KCM" cell line; see Figure 3) generated from the triple transgenic PDA.MUC1 .Tg mice. While applicants do not wish to be bound by any particular theory of operation, it appeared that the antibody activated natural killer cells (NK cells) and conjugation with CpG ODN further enhanced the NK cell lytic activity against its targets such as YAC cells as well as the KCM cells lines (see Figure 4).
  • NK cells natural killer cells
  • YAC cells as well as the KCM cells lines
  • mice Ten (10) mice were injected with the KCM established pancreatic cancer cell line generated from the triple transgenic PDA.MUC1 .Tg mice.
  • the forward primers were identical to the ones used in the first-round RT-PCR, although the amount of each primer was doubled relative to the RT-PCR conditions described above.
  • Semi-nested reverse primers specific for heavy chain sequecnes were used at 100 ng per reaction.
  • the PCR conditions employed were as follows:
  • the plasmids from were transfected into CHO cells, and recombinant IgG having a human lgG1 backbone were produced.
  • Supernatants from transfected CHO cells were tested for antigen binding in 96-well ELISA format.
  • concentrations of the recombinant IgG in the supernatants were low (i.e., about 10 ng/ml).
  • the ELISA results indicated that the recombinant antibodies were very potent.
  • sequences of the inserts of the recombinant antibody plasmids were determined by DNA sequencing. All corresponded to a single heavy chain sequence and a single light chain sequence.
  • the nucleotide and amino acid sequences of the variable regions of the heavy and light chains were determined, and the CDR sequences deduced therefrom were as set forth in SEQ ID NOs: 8-13.
  • TAB-004 To that end, the ability of TAB-004 to detect levels of shed MUC1 in the plasma of patients was compared to the performances of these antibodies in plasma samples.
  • An enzyme immunoassay (EIA) has been optimized using the TAB-004 antibody to capture and detect levels of shed MUC1 present in circulation. Briefly, a 96-well ELISA plate was coated with 100 ⁇ of TAB-004 antibody at 50 ⁇ 9/ ⁇ _ in PBS and incubated overnight at 4°C. Following incubation, excess TAB-004 capture antibody was removed from the plate. The plate was blocked with 200 ⁇ of 1 % dry milk in PBS to avoid non-specific binding and incubated for 1 hour at 4°C.
  • the plates were washed extensively (3 times) with 250 ⁇ PBS containing 0.05% (v/v) TWEEN®-20 using an ELISA plate washer.
  • a MUC1 standard using a 25-mer polypeptide preparation from MUC1 TR was prepared ranging from 0-2000 U/ml.
  • the test plasma was diluted 1 :2, and 1 :10 in 0.1 % milk in PBS, and 100 ⁇ ws added to the appropriate wells in triplicate, and the plates were incubated for 2 hours at 37°C.
  • the reaction was stopped by the addition of 25 ⁇ of 4.0 N sulfuric acid, and the optical density read at a wavelength of 450 nm using a SPECTRA-MAX 250 spectrophotometer. Standard curves were generated with regression analyses to determine concentrations of the unknown samples.
  • Arbitrary units (U)/ml were chosen based on an initial reference standard of MUC1 .
  • a linear range was determined to be 50-800 units/ml of MUC1 antigen.
  • Intra-and inter-assay variations were controlled by including normal and abnormal samples to ensure the equipment, the technologist, and the reagents used in the test were performing as expected.
  • the TAB-004 EIA assay was compared against CA15-3 (Abbott Laboratories, Abbott Park, Illinois, United States of America) breast cancer samples. Comparison to CA27-29 (Bayer Diagnostics, Tarrytown, NY) for breast cancer and to CA19-9 (Panomics Inc, Redwood City, California, United States of America) for pancreatic cancer samples using EIA assays are also performed. Statistical analyses were conducted to assess the sensitivity and specificity of TAB-004 EIA versus the commercially available EIAs.
  • the TAB-004 EIA was employed using plasma from 36 breast cancer patients, 24 prostate cancer patients, 4 pancreatic cancer patients, 13 esophageal cancer patients, 12 normal controls, 3 patients with pancreatitis, and 1 diabetic patient (pancreatitis and diabetes are two conditions known to detect as falsely positive using the assays that test for the presence of the CA 15-3, CA 27-29, and CA 19-9 antigens). Most cancer samples that were tested were from late stage cancers.
  • the TAB-004 assay provided values that were higher than CA 15-3 for all 20 samples. Further, the TAB-004 assay levels were dependent on tumor stage, as they increased with disease progression (p ⁇ 0.0001 ) unlike the CA 15-3 assay, which was unable to predict differences between stages 0, 2, and 3.
  • TAB-004 was superior to CA 15-3 for diagnosing stage 2 and 3, as all stage 2 and stage 3 patients displayed TAB-004 assay values above the normal range while only 2 out of 10 were above normal for the CA 15-3 assay.
  • TAB-004 is employed to assess its potential in accurately predicting disease recurrence and progression.
  • Plasma from n 100 patients at stages II and lll/IV breast cancer are assessed pre-treatment and 6, 12, and 18- months post end of standard of care therapy. Recurrence in this group is assessed after 24 months.
  • Plasma from n 50 pancreatic patients at stages 2, 3, and 4 is assessed pre-treatment, and 3, 6, 9, and 12 months post standard of care therapy.
  • Plasma Plasma is collected at routine follow-up visits.
  • the recurrence rate is generally much lower compared to pancreatic cancer, and therefore lower numbers of pancreas cancer patients (statistically justified below) are employed.
  • the times for collecting plasma also differ between breast and pancreas cancer due to standard follow-up visit timing.
  • stage 3 and 4 patients For pancreatic cancer patients, few stage 3 and 4 patients generally survive more than 6 months to a year, and thus it is not possible to wait for end of treatment to collect samples again due to the high mortality rate after diagnosis.
  • the samples are collected while the patients are undergoing therapy. For patients that undergo surgery, samples are collected before surgery and 3, 6, 9, and 12 months post surgery regardless of their treatment regimen. For patients that have un-resectable cancer, samples are collected at diagnosis prior to start of therapy and then at 3, 6, 9, 12 months post diagnosis regardless of the treatment regimen. Since most patients would be expected to recur within a year, the trial is stopped at 12 months. For breast cancer patients, only stage lll/IV are generally expected to recur within 2 years. However for comparison, stage II patients are included. Plasma is collected pre-treatment and then at 6, 12, and 18 months post end of treatment.
  • ROCs Receiver Operating Curves
  • Cox proportional hazard models are performed with time to recurrence as the dependent (outcome) variable.
  • the Cox model is a multivariate procedure that correctly accounts for lost to follow-up and censored data. Age, cancer stage, and cancer grade, and TAB- 004 values are entered as independent predictors. If the ROC determines a natural cut-point for predicting recurrence, then another Cox model is run with this dichotomous (above or below the cutoff) variable replacing the actual value of TAB-004 in the model.
  • TAB-004 is an independent predictor of recurrence when adjusting for the patient's age, cancer stage, and grade of the tumor.
  • the previous set of analyses is repeated with time to recurrence or death as the dependent variable.
  • stage lll/IV metalastatic
  • This set of analyses is also performed using the data from the patients with pancreatic cancer. Wth the extremely high death rate for pancreatic cancer, getting stable estimates of the coefficients in the Cox model when predicting time to recurrence or time to stage 4 disease can be difficult. As such, few recurrences of cancer or few cases of patients progressing from stages 2 or 3 to stage 4 metastatic cancers might occur during the study period.
  • Circulating Tumor Cells are defined as tumor cells in the bloodstream.
  • the Veridex CELLSEARCH® system is the only FDA approved method to measure CTCs in metastatic breast, colorectal and prostate cancer patients.
  • CTCs in pancreatic cancer patients have shown a correlation between CTCs ⁇ 1 and survival.
  • the presence of CTCs was shown to correlate with increased sera levels of the tumor antigen CA 19-9, indicating that sera levels of tumor antigens can also be predictive of tumor cells in circulation.
  • ⁇ 5 CTCs was an independent predictor of progression-free survival and overall survival. Further, CTC levels in metastatic breast cancer patients are an earlier, more reproducible indication of disease status than current imaging methods.
  • the approved method for CTC assessment entails isolating EpCAM- expressing cells from the blood and then validating these cells as CTCs with the presence of epithelial-specific cytokeratin staining, proper nucleus staining, and the absence of leukocyte-specific CD45.
  • a caveat to this method is the restriction to EpCAM expressing cells. Studies have suggested that cells acquiring a migratory phenotype lose their epithelial characteristics and acquire mesenchymal features, which phenotypically have been shown to be the cells responsible for aggressive tumor progression. As such, it is apparent that EpCAM isolation of CTCs could "miss" the most potent CTCs - those possessing a mesenchymal phenotype.
  • TAB-004 antibody Both breast and pancreatic primary and metastastatic tumors express high levels of tumor-associated MUC1 recognized by our TAB-004 antibody. Therefore, CTCs in these patients should be recognized by the TAB-004 qntibody. In preliminary experiments, levels of MUC1 were assessed using the TAB-004 antibody.
  • the ability to use the Veridex CELLSEARCH® system to measure MUC1 -expressing CTCs using 7.5 ml blood samples (analogous to human samples) spiked with PANC1 cells, which are a human pancreatic cancer cell line was tested. Approximately 90% of the CTCs (EpCAM+ cells) expressed MUC1 . Further, patient samples were collected, and it was found that TAB-004 recognizes CTCs at from about 33% to 100% efficiency. Therefore, using the TAB-004 antibody appears to accurately detect micrometastases in pancreatic and breast cancer patients.
  • NM_004985.3 NP_001 181906; NP_004976; NP_036734; NP_038633;
  • SEQ ID NO : 1 MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRS SVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTS VPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPP AHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTR PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD
  • SEQ ID NO: 2 MTEYKLWVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQ WIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQI KRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFY TLVREIRKHKEKMSKDGKKKKKKSKTKCVIM SEQ ID NO: 3: STAPPVHNVTSAPDTRPAPGSTAPP
  • SEQ ID NO: 5 EVQLQQSGGERATPGASVKMSCKTSGYTFTNYWMHWVKQRPGQ GLEWIGYINPSSGYTQYNQKFKDKATLTADKSSSTAYIQLSSLTSEDSAVYYCSTYY GDYLFPYWGQGTLVTVSA
  • SEQ ID NO : 7 DVLMTQTPLSLPVSLGDQASISCRSSQDIVYGNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP YTFGGGTKLEIKR

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Abstract

La présente invention concerne des anticorps isolés, et des fragments et dérivés de ceux-ci, qui se lient à des antigènes tumoraux. La présente invention concerne en outre des compositions et des agents d'administration qui comprennent les anticorps décrits et des fragments et dérivés de ceux-ci; des cellules qui produisent ceux-ci; des procédés pour produire ceux-ci; des procédés d'utilisation de ceux-ci pour détecter, cibler, et/ou traiter des tumeurs et/ou des cellules métastatiques dérivés de celles-ci et/ou des cellules souches tumorales; et des procédés pour prédire la récurrence de cancer chez un sujet.
PCT/US2011/037972 2009-10-08 2011-05-25 Anticorps spécifiques à une tumeur et utilisations de ceux-ci WO2012047317A2 (fr)

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AU2011312830A AU2011312830B2 (en) 2010-10-08 2011-05-25 Tumor specific antibodies and uses therefor
CN201180059040.8A CN103492417B (zh) 2010-10-08 2011-05-25 肿瘤特异的抗体和其用途
KR1020137011650A KR101883280B1 (ko) 2010-10-08 2011-05-25 종양 특이 항체 및 그 사용
BR112013008480A BR112013008480A2 (pt) 2010-10-08 2011-05-25 anticorpos contra tumor e usos específicos dos mesmos
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CA2813814A CA2813814C (fr) 2010-10-08 2011-05-25 Anticorps specifiques a une tumeur et utilisations de ceux-ci
RU2013120483/10A RU2595403C2 (ru) 2010-10-08 2011-05-25 Специфические к опухоли антитела и их применение
DK11831066.3T DK2624866T3 (da) 2010-10-08 2011-05-25 Tumorspecifikke antistoffer og anvendelser deraf
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IL225545A IL225545A0 (en) 2010-10-08 2013-04-03 Antibodies specific to cancerous tumors and their uses
US14/810,209 US20150368356A1 (en) 2009-10-08 2015-07-27 Tumor specific antibodies and uses therefor
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WO2017030506A1 (fr) * 2015-08-18 2017-02-23 Agency For Science, Technology And Research Procédé de détection de cellules tumorales circulantes et ses utilisations
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US9845362B2 (en) 2010-10-08 2017-12-19 The University Of North Carolina At Charlotte Compositions comprising chimeric antigen receptors, T cells comprising the same, and methods of using the same
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WO2014047278A1 (fr) * 2012-09-20 2014-03-27 Viracor-Ibt Laboratories Procédé de détermination de la réponse immunitaire cellulaire aux vaccins thérapeutiques
WO2016090434A1 (fr) * 2014-12-12 2016-06-16 Commonwealth Scientific And Industrial Research Organisation Délogement et libération des csh à l'aide d'un antagoniste de l'intégrine alpha 9 et d'un antagoniste de cxcr4
WO2017030506A1 (fr) * 2015-08-18 2017-02-23 Agency For Science, Technology And Research Procédé de détection de cellules tumorales circulantes et ses utilisations
US10111966B2 (en) 2016-06-17 2018-10-30 Magenta Therapeutics, Inc. Methods for the depletion of CD117+ cells
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WO2020221849A1 (fr) * 2019-05-01 2020-11-05 Universidade De Santiago De Compostela Administration intracellulaire d'anticorps anti-kras formulés dans des nanocapsules

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